Acetochlor is a preemergent chloroacetanilide herbicide used to control annual grasses and small-seeded broadleaf weeds. It is the second most abundantly applied herbicide on corn crops in the United States; however, human metabolites associated with known exposure to acetochlor have not been positively identified and confirmed. We positively identified acetochlor mercapturate (ACM) as a metabolite of acetochlor in urine samples collected during a 24-h period from custom (commercial) applicators who had applied acetochlor on either the day of or the day before urine collection. Concentrations in applicator urine samples ranged from 0.5 to 449 mg/l (0.3-121 mg/g creatinine). We found that ACM accounted for as much as 42% of the total acetochlor-derived metabolites; however, as the exposure level decreased (based on total acetochlor metabolite level), ACM became a less abundant metabolite of acetochlor (o17%). Unmetabolized acetochlor was also measured in the urine samples analyzed. At high exposures (classified as 4100 mg/l), acetochlor accounted for about 0.8% of the total excreted acetochlor metabolites (B2% of the ACM concentrations). At lower exposures (classified as ACM o10 mg/l), ACM and acetochlor concentrations were similar. Additionally, we tentatively identified another acetochlor metabolite that appeared to be important at low levels of exposure.