Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is among the top 100 drugs prescribed annually in the United States and the United Kingdom and is one of many pharmaceutical products that have been detected in global surface waters. Our study used sublethal concentrations to assess the impact of FLX exposure on sheepshead minnow (Cyprinodon variegatus) locomotor behavior. Exposures lasted for 56 hours, and fish were recorded for locomotor behavior assessment at six timepoints between 1-56 h post-dose. Behavior was recorded to quantify locomotor activity using line crossing counts. Animals treated with 300 μg L(-1) FLX exhibited reduced locomotor activity at 1, 25, 32, 49 and 56 h post-dose. An EC(25) value of 2 μg L(-1) (lower and upper 95 % confidence limits at 1.3, 43 μg L(-1), respectively) was determined for locomotor activity at 32 h of exposure. Changes in locomotor activity due to FLX exposure may have implications for the ecological response of populations to other natural and anthropogenic stressors.