The activities of cholinesterase (ChE) and glutathione S-transferase (GST) enzymes were assessed in the wolf spider (Lycosa hilaris) as biomarkers of organophosphate contamination in agricultural ecosystems. Spiders were exposed to simulated field rates of two commercially available organophosphorous insecticides [Basudin (diazinon) and Lorsban (chlorpyrifos)] under laboratory conditions. In terms of survival, chlorpyrifos and diazinon were more toxic to male than to female wolf spiders, but gender-specific differences in ChE activities were not evident. Cholinesterase activity in male spiders was inhibited to 14% and 61% of control activity by Basudin and Lorsban, respectively. Gluthathione S-transferase activity was not affected by either pesticide. Mortality and biomarker responses in the wolf spider were further investigated following the application of Basudin to pasture. Wolf spiders were deployed into field mesocosms; after 24 h mortality was 40%, and surviving spiders displayed significant inhibition of ChE activity (87%) compared with controls. Cholinesterase activity in spiders exposed for subsequent 24- or 48-h time periods was monitored until it returned to control levels 8 days post-application. Inhibition of ChE activity after a single application of Basudin indicate the potential use of this enzyme in wolf spiders as a biomarker for evaluating organophosphate contamination.