The use of chemical inhibitors/inducers is one of the strategies employed to determine whether a particular metabolic pathway is involved in the metabolism of a xenobiotic. The objective of this study was to assess the role of piperonyl butoxide (PBO) on the toxicity of an organophosphorus insecticide, chlorpyrifos (CPF) to two species, Ceriodaphnia dubia (waterflea) and Xenopus laevis (South African clawed frog). Chlorpyrifos was highly toxic to C. dubia (48-h LC50: 0.05 microg/L) in comparison with X. laevis (96-h LC50: 2410 microg/L). Piperonyl butoxide at 200 microg/L reduced the toxicity of chlorpyrifos to C. dubia by a factor of 6. Piperonyl butoxide at 3000 microg/L also reduced the toxicity of CPF to X. laevis with respect to mortality and malformations. Acetylcholinesterase (AChE) activity was used as a biomarker to further assess the role of PBO in chlorpyrifos toxicity. X. laevis exposed to CPF and PBO exhibited a biphasic response in terms of AChE activity with an initial increase in the AChE activity followed by a drastic decrease. The results from the present study indicate that C. dubia and X. laevis have the capability to metabolize chlorpyrifos via cytochromes P450 mediated reactions. The results also indicate that the use of the biomarker AChE is useful in determining metabolic processes of organophosphorus insecticides, which require metabolic activation.