Drug interactions have been suggested as a cause of Gulf War Syndrome. Pyridostigmine bromide (PB), a prophylactic treatment against potential nerve gas attack, the insect repellent DEET, and permethrin (PERM) impregnated in soldiers' uniforms may have interacted and caused greater than expected toxicity. We tested those 3 drugs singly and in combinations on male and female Sprague-Dawley rats in open field arenas to find the effects on rate of locomotion and thigmotaxis. Administration rates were 10 mg PB/kg; 50, 200, or 500 mg DEET/kg; 15, 30, or 60 mg PERM/kg; 5 mg PB/kg + 100 mg DEET/kg; 5 mg PB/kg + 15 mg PERM/kg; 100 mg DEET/kg + 15 mg PERM/kg; or vehicle by gavage and i.p. injection. Locomotor behavior was quantified by video-computer analysis for 2 h post-treatment. Female rats were tested in either pro- or metestrus. Drug interactions were determined by the isobolographic method. Blood serum drug concentrations were estimated by high performance liquid chromatography or gas chromatography-mass spectrometry. Single drug effects were very limited within the ranges tested. Double-drug administrations at half the single-drug rates resulted in statistically significant interactions in male rats for both locomotion rate and thigmotaxis. Combination of PB + PERM and DEET + PERM significantly affected speed, whereas only the combination of DEET + PERM significantly affected thigmotaxis. Female rats did not show significant interactions. Our data suggest that serum concentrations of PB and DEET may have been higher in females than males. Administration of PB + DEET may have reduced the serum concentration of DEET, and administration of PB + PERM may have increased the serum concentration of PERM.