The purpose of this study was to assess intraspecies differences in behavioral and autonomic function in three strains of rat following administration of diisopropyl fluorophosphate (DFP), an irreversible inhibitor of acetylcholinesterase activity. Male rats of the Long‐Evans (LE), Fischer 344 (F344), and Sprague‐Dawley (SD) strains were administered DFP at doses of 0–1.5 mg/kg (sc). The animals were placed 60 min later into one of two motor activity chambers and tested for 30 min. Motor activity was measured using either a Doppler‐based system or a commercial photocell device. Following measurement of motor activity in the Doppler system, body temperature (Tb) was measured and blood was then withdrawn by cardiac puncture and analyzed for serum cholinesterase activity (ChE). The remaining rats were retested 1 d after DFP administration in the photocell device. The results showed a significant influence of strain on the effects of DFP. Motor activity of LE rats was reduced by DFP at doses of 1.0 and 1.5 mg/kg, whereas the activity of F344 rats was reduced only at 1.5 mg/kg. The relative sensitivity of SD rats depended on the device used to measure motor activity. The SD rats resembled F344 rats in their response to DFP when motor activity was measured in the photocell device, and LE rats when motor activity was measured in the Doppler system. The Tbof F344 rats was unaffected by DFP, while the LE and SD rats became hypothermic at 1.5 mg/kg. The DFP‐induced inhibition of serum ChE activity was significantly less in F344 rats. All three strains retested the day after DFP still showed significant decreases in motor activity. Overall, it appears that the F344 strain is relatively resistant to the behavioral and autonomic effects of DFP. This intraspecies variability should be considered in selecting appropriate experimental models for assessing the neurotoxicological hazards of cholinesterase‐inhibiting pesticides.