A study of the Withlacoochee River watershed in west-central Florida was conducted from October 2003 to March 2007 to gain a better understanding of the hydrology and surface-water and groundwater interactions along the river. The Withlacoochee River originates in the Green Swamp area in north-central Polk County and flows northerly through seven counties, emptying into the Gulf of Mexico. This study includes only the part of the watershed located between the headwaters in the Green Swamp and the U.S. Geological Survey gaging station near Holder, Florida. The Withlacoochee River within the study area is about 108 miles long and drains about 1,820 square miles. The Withlacoochee River watershed is underlain by thick sequences of carbonate rock that are covered by thin surficial deposits of unconsolidated sand and sandy clay. The clay layer is breached in many places because of the karst nature of the underlying limestone, and the degree of confinement between the Upper Florida aquifer and the surficial aquifer is highly variable throughout the watershed. The potential for movement of water from the surface or shallow deposits to deeper deposits, or from deeper deposits to the shallow deposits, exists throughout the Withlacoochee River watershed. Water levels were higher in deeper Upper Floridan aquifer wells than in shallow Upper Floridan aquifer wells or surficial aquifer wells at 11 of 19 paired or nested well sites, indicating potential for discharge to the surface-water system. Water levels were higher in shallow Upper Floridan aquifer or surficial aquifer wells than in deeper Upper Floridan aquifer wells at five other sites, indicating potential for recharge to the deeper Upper Floridan aquifer. Water levels in the surficial aquifer and Upper Floridan aquifer wells at the remaining three sites were virtually the same, indicating little or no confinement at the sites. Potentiometric-surface maps of the Upper Floridan aquifer indicate the pattern of groundwater flow in the aquifer did not vary greatly from season to season during the study. Potentiometric contours indicate groundwater discharge to the river in the vicinity of Dade City and Lake Panasoffkee. During dry periods, groundwater from the underlying Upper Floridan aquifer contributed to the flow in the river. During wet periods, streamflow had additional contributions from runoff and input from tributaries. Groundwater has a greater effect on streamflow downstream from the Dade City station than upstream from the Dade City station because confinement between surficial deposits and the Upper Floridan aquifer is greater in the Green Swamp area than in downstream areas. Estimates of streamflow gains and losses were made along the Withlacoochee River during base-flow conditions in May 2004, April 2005, and April 2006. Base flow was higher in April 2005 than in May 2004 and April 2006. Consistent net seepage gains were identified in 16 of 20 subreaches analyzed during all seepage runs. The direction of exchange was variable in the remaining four subreaches. Low specific conductance, pH, and calcium concentrations in water from the Withlacoochee River near the headwater area indicated a surface-water system not directly connected to the Upper Floridan aquifer. Downstream from the Dade City station, higher specific conductance, pH, and calcium concentrations in the river water indicated an increasing influence of groundwater, and were similar to groundwater during low-flow conditions. Strontium isotope ratios indicate groundwater originates from shallow parts of the Upper Floridan aquifer in the upper reaches of the river, and from increasingly deeper parts of the aquifer in the downstream direction. Mean annual base-flow estimates also indicate increasing groundwater discharge to the river in the downstream direction. Mean annual base flow estimated using standard hydrograph separation method assumptions ranged from about 4.7 to 5.1 inches per year