We studied diel movement and habitat use by paddlefish Polyodon spathula implanted with radio transmitters in Navigation Pool 8 of the upper Mississippi River. We radio-tracked five paddlefish during three randomly chosen 24-h periods each month in May, Aug, and Oct 1995. Paddlefish were located by boat one to three times every 3 h during each 24-h period. At each location, geographic coordinates were determined with a global positioning system receiver using the Precise Positioning Service, and depth was measured with a depth sounder. Location coordinates were plotted with ARC/INFO software on a Geographic Information System land–water coverage. Movement distances were calculated as the linear distance between sequential locations. Radio-tagged paddlefish usually remained in a secondary channel that had low current velocity during all seasons, whereas main channel, main channel border, tailwater, and backwater habitats were seldom used. Paddlefish strongly selected areas that were deep; about 62% of paddlefish locations were in areas with more than 6 m of depth, although this habitat constituted only 14.5% of the total study area. However, paddlefish used significantly shallower areas during the night than during the day. Paddlefish moved significantly larger distances at night than during the day in spring and fall, but differences in movement among diel periods during summer were not significant. Our research suggests that radiotelemetry studies that need to determine depth use or movement of paddlefish during small time scales may need to incorporate a diel component. However, study objectives to determine use of general habitat types by radio-marked paddlefish can be adequately met by tracking during the day.