Abstract-The potential use of cholinesterase (ChE) in body muscle of three-spined stickleback (Gasterosteus aculeatus) as a biomarker of organophosphate (OP) contamination was investigated. Fish were sampled from nine headwater streams directly after the main application period (July 1995) and 2 months after the end of OP use (October 1995). Cholinesterase activity was inversely related to fish weight. Muscle ChE was comprised of both acetylcholinesterase and pseudocholinesterase. In July, fish from two sites (A and B) had significantly lower (ϳ30%) ChE activities than fish from all but one of the other sites. In October, the range of ChE activities was similar to that observed in July; however, no site significantly differed from the majority of other sites. The OP parathion was detected in suspended particulates at sites A and B. This suggests that decreased ChE in July reflects OP exposure. Because sampling of suspended particulates at site A occurred with a higher resolution than at the other sites and because decreases were close to the natural variability, however, this relationship cannot be demonstrated with certainty. The reported characteristics of ChE and its natural variability in stickleback represent baseline data for its use in the monitoring of OP contamination of small streams.