Pretreatment of rats with the nonspecific esterase inhibitor iso‐OMPA (1 mg/kg sc) 1 h prior to carbofuran (2,3‐dihydro‐2,2‐dimethyl‐7‐benzofuranylN‐methylcarbamate, 0.5 mg/kg sc) administration potentiated carbofuran toxicity by more than threefold. Neither iso‐OMPA nor carbofuran in the given doses produced any gross toxic signs. Rats receiving combined treatment, however, showed severe hypercholinergic signs (salivation, tremors, muscle fasciculations, and convulsions) within 5–10 min following carbofuran administration, and the severity was comparatively greater than that observed with an acute dose of carbofuran (1.5 mg/kg sc). Rats pretreated with iso‐OMPA (0.5 mg/kg) died within 10–15 min following the acute dose of carbofuran (1.5 mg/kg). Each drug when given alone (1.0 mg/kg iso‐OMPA, 0.5 mg/kg carbofuran) caused a significant (p< .01) inhibition of carboxylesterase (CarbE) activity in brain structures (cortex, stem, striatum, and hippocampus), skeletal muscle (hemi‐diaphragm), liver, and plasma, whereas acetylcholinesterase (AChE) activity remained significantly (p> .01) unchanged. The maximal CarbE inactivation in plasma (< 14% remaining activity) following either drug indicated a tremendous nonspecific binding to non‐AChE serine‐containing enzymes. iso‐OMPA pretreatment markedly potentiated carbofuran's anticholinesterase activity both in neuronal and in nonneuronal tissues. It can be concluded that iso‐OMPA pretreatment potentiates carbofuran toxicity either by preventing nonspecific binding of carbofuran to CarbE and/or possibly by inhibiting its detoxification.