The toxic effect of sublethal concentrations (1, 17.5 and 35 mg L(-1)) of pure glyphosate was evaluated on acetylcholinesterase (AChE) activity in the fish species, Cnesterodon decemmaculatus. Acute bioassays (96 h) under laboratory conditions were conducted and homogenates for each specimen corresponding to the anterior, middle and posterior body sections were performed. Fish survival was 100%, even at the highest concentration tested (35 mg L(-1)), in accordance with the low lethal toxicity reported for glyphosate. However, a significant inhibitory effect on AChE activity was recorded even for the lowest herbicide concentration tested (1 mg L(-1)), in the homogenates corresponding to the anterior body section. The inhibition ranged from 23 to 36%. The analytical determination of glyphosate in assay media by ion chromatography, was used to verify its stability. These results indicate that AChE-a neurotoxicity biomarker-in C. decemmaculatus may be affected by exposure to environmentally relevant concentrations of glyphosate.