The metabolism of butachlor was studied in rat liver and kidney homogenates. In vitro incubation of butachlor with liver fractions (S9, microsome, and cytosolic fractions) formed a considerable amount of butachlor glutathione conjugate (BGSC), while the conjugating activity was not efficient for the kidney S9 fraction. There is a sex difference in the distribution of glutathione S‐transferase in the liver. It seems that more enzyme activity is detected in the female liver microsome, while this is not the case in its cytosolic fraction. Further biotransformation of BGSC to mercapturate was not observed in the liver S9 fraction. This metabolite was further transformed to butachlor acetyl cysteine conjugate (BACC) in the presence of acetyl CoA, but to buta‐ chlor cysteine conjugate (BCC) in the absence of acetyl CoA. These findings demonstrated that butachlor is initially conjugated with CSH to form BGSC by the enzyme glutathione S‐transferase in the liver. This metabolite is apparently transported to the kidneys, where it is transformed to the mercapturate.