Steelhead trout (Oncorhynchus mykiss) were exposed for 96 h to the organophosphate chlorpyrifos to establish benchmark concentration (BMC) values in the low-effect range of brain acetylcholinesterase (AChE) inhibition. The U.S. Environmental Protection Agency (U.S. EPA) benchmark dose software was used to model the data. Benchmark concentrations were determined for a range of inhibition levels at 5, 10, and 20%, at 1 and 2 control standard deviations (SD), and at an experimental limit-of-detection level of 2.5%. One contributing difficulty in establishing precise inhibition BMCs in the lower effect region is the variability associated with the AChE analytical method. To minimize this variability, the Ellman method was modified specifically for analysis of O. mykiss brain tissue. Laboratory-established BMCs for chlorpyrifos were then compared with the U.S. EPA 96-h water quality criteria and with the concentration levels detected in Northwest surface waters that are home to threatened steelhead trout. The U.S. EPA 96-h water quality criteria of 0.083 microg/L is below the BMC(02.5), the limit-of-detection value for this study. The average chlorpyrifos concentration detected during a two-week period in one monitored stream was 0.127 microg/L, which approaches the BMC(02.5). The peak chlorpyrifos concentration detected at 0.482 microg/L is near the BMC(1SD) estimate.