Many karst regions are undergoing rapid population growth and expansion of urban land accompanied by increases in wastewater generation and changing patterns of nitrate (NO₃⁻) loading to surface and groundwater. We investigate variability and sources of NO₃⁻ in a regional karst aquifer system, the Edwards aquifer of central Texas. Samples from streams recharging the aquifer, groundwater wells, and springs were collected during 2008–12 from the Barton Springs and San Antonio segments of the Edwards aquifer and analyzed for nitrogen (N) species concentrations and NO₃⁻ stable isotopes (δ¹⁵N and δ¹⁸O). These data were augmented by historical data collected from 1937 to 2007. NO₃⁻ concentrations and discharge data indicate that short-term variability (days to months) in groundwater NO₃⁻ concentrations in the Barton Springs segment is controlled by occurrence of individual storms and multi-annual wet-dry cycles, whereas the lack of short-term variability in groundwater in the San Antonio segment indicates the dominance of transport along regional flow paths. In both segments, longer-term increases (years to decades) in NO₃⁻ concentrations cannot be attributed to hydrologic conditions; rather, isotopic ratios and land-use change indicate that septic systems and land application of treated wastewater might be the source of increased loading of NO₃⁻. These results highlight the vulnerability of karst aquifers to NO₃⁻ contamination from urban wastewater. An analysis of N-species loading in recharge and discharge for the Barton Springs segment during 2008–10 indicates an overall mass balance in total N, but recharge contains higher concentrations of organic N and lower concentrations of NO₃⁻than does discharge, consistent with nitrification of organic N within the aquifer and consumption of dissolved oxygen. This study demonstrates that subaqueous nitrification of organic N in the aquifer, as opposed to in soils, might be a previously unrecognized source of NO₃⁻ to karst groundwater or other oxic groundwater systems.