This study investigated the catalytic activities of hepatic glutathione S-transferase (GST) isoenzymes isolated from CD-1 mice toward two activated alkenals of toxicological relevance: trans,trans-muconaldehyde (MA), a putative myelotoxic metabolite of benzene, and trans-4-hydroxy-2-nonenal (HNE), a highly reactive lipid peroxidation product. The activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was also determined. Four isoenzymes with pI values of 9.8, 8.7, 6.4, and 5.7 were each isolated from male and female mice. The isoenzymes with pI values of 8.7 and 6.4 are pi and mu class GSTs, respectively, whereas the pI 9.8 and 5.7 GSTs are both alpha class isoenzymes. CDNB activity was greatest in the pi (pI 8.7) isoenzyme of both sexes. In addition, the CDNB activity of the pi (pI 8.7) isoenzyme from males was markedly greater than the corresponding GST from female mouse liver. In contrast to CDNB, both MA and HNE were better substrates for the acidic alpha (pI 5.7) and mu (pI 6.4) GSTs, whereas minimal activity toward either alkenal was detected in the pi (pI 8.7) and alpha (pI 9.8) isoenzymes. Maximum activity toward MA and HNE was exhibited by the alpha (pI 5.7) isoenzyme of both sexes. The level of HNE activity observed with the alpha (pI 5.7) isoenzyme was five- to sixfold greater than that reported previously for any mouse GST isoenzyme. Moreover, the specific activities of the female alpha (pI 5.7) isoenzyme toward both HNE and MA were markedly greater than those of the corresponding isoenzyme from males. A similar gender-specific difference was noted in the activity of the mu (pI 6.4) isoenzyme toward HNE, but not toward MA. These results show that both MA and HNE are substrates for the alpha (pI 5.7) and mu (pI 6.4) GSTs of murine liver, with maximum activity toward both activated alkenals exhibited by the alpha (pI 5.7) isozyme. In addition, evidence is presented that demonstrates a female-dominant sex difference in the activity of the alpha (pI 5.7) isoenzyme toward MA and HNE, which contrasts sharply with the male-dominant activity of pi class GSTs toward CDNB. These results are consistent with the hypothesis that alpha and mu class GSTs are critical detoxication enzymes in female mouse liver, whereas pi-class GST isozymes predominate in the liver of male mice.