Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides for the control of the horn fly, Haematobia irritans (L.) in many countries. PBO-synergized organophosphate (OP) insecticides are also available for horn fly control in the United States. PBO is known to inhibit the bio-activation of certain OPs including diazinon. A study was conducted to evaluate the effects of PBO on toxicity of diazinon to horn flies using a filter paper bioassay technique. Results from this study indicated a biphasic effect of PBO on diazinon toxicity to the horn fly. PBO inhibited diazinon toxicity when the concentration of PBO used was higher (5%), and no effect was observed when PBO concentration was intermediate (2%). At lower concentrations (1% and lower), PBO significantly synergized diazinon toxicity. Enhanced esterase activity was found to be associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases suggest that PBO did not have significant effect on esterase activity in the horn fly. Therefore, the observed synergistic effect of PBO at lower concentrations (1% and lower) on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. We hypothesized that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the fly cuticle, while inhibiting diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.