This study assessed the in vitro and in vivo effects of an acetylcholinesterase enzyme inhibitor (chlorpyrifos) in two estuarine crustaceans: grass shrimp (Palaemonetes pugio) and mysid (Americamysis bahia). The differences in response were quantified after lethal and sublethal exposures to chlorpyrifos and in vitro assays with chlorpyrifos-oxon. Results from the in vitro experiments indicated that the target enzyme, acetylcholinesterase (AChE), in the two species was similar in sensitivity to chlorpyrifos inhibition with IC50s of 0.98 nM and 0.89 nM for grass shrimp and mysids, respectively. In vivo experiments showed that mysids were significantly more sensitive to chlorpyrifos-induced AChE inhibition after 24 h of exposure. The in vivo EC50s for AChE inhibition were 1.23 μg L(-1) for grass shrimp and 0.027 μg L(-1) for mysids. Median lethal concentrations (24h LC50 values) were 1.06 μg L(-1) for grass shrimp and 0.068 μg L(-1) for mysids. The results suggest that differences in the response of these two crustaceans are likely related to differences in uptake and metabolism rather than target site sensitivity.