Chlorpyrifos is a commonly used insecticide that can be metabolically activated by CYP2B to the acetylcholinesterase inhibitor chlorpyrifos-oxon causing cholinergic overstimulation and neurotoxicity. Rat brain extracts can also activate chlorpyrifos in vitro, and the lack of circulating oxon in serum suggests that metabolic activation within the brain may be responsible for chlorpyrifos neurotoxicity. Rats received intracerebroventricular (ICV) injections of CYP2B mechanism-based inhibitors (MBI), once or repeatedly, followed by chlorpyrifos (62.5-250 mg/kg sc). Rats were assessed for neurochemical (acetylcholinesterase activity), physiological (temperature), and behavioral measures (e.g., gait, righting reflex, arousal, incline angles) at 4 hours 3 days after chlorpyrifos treatment. ICV CYP2B MBIs increased brain chlorpyrifos levels, decreased brain chlorpyrifos-oxon levels, and attenuated the reduction in brain acetylcholinesterase; there was no effect on serum chlorpyrifos levels or acetylcholinesterase activity reduction. Inhibition of brain chlorpyrifos metabolism by CYP2B MBIs blocked centrally mediated hypothermia but not peripherally mediated hyperthermia. A single ICV MBI treatment significantly attenuated chlorpyrifos neurotoxicity mediated behavioral outcomes at 1 day after chlorpyrifos treatment with a gradual worsening of behavioral scores through day 3, suggesting a recovery of brain CYP2B activity and an increase in local chlorpyrifos activation. Daily ICV MBI injections attenuated neurotoxicity across all test days consistent with prolonged inhibition of brain chlorpyrifos activation. Thus, rat brain CYP2B contributes significantly to chlorpyrifos's neurotoxic effects. Variable human brain CYP2B levels, influenced by genetics and environmental exposures, may contribute to interindividual differences in neurotoxicity. Therapeutic inhibition of brain CYP2B could also be explored as a treatment for exposure to CYP2B-activated neurotoxins.