The present study describes cDNA cloning, expression, and kinetic characterization of the two subunits of a murine alpha-class glutathione (GSH) S-transferase (GST) isoenzyme (previously designated as GST 9.5), which, unlike other alpha-class mammalian GSTs, is exceptionally efficient in the GSH conjugation of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-anti-BPDE] [X. Hu, S. K. Srivastava, H. Xia, Y. C. Awasthi, and S. V. Singh (1996) J. Biol. Chem. 271, 32684-32688]. The cDNAs for both subunits of GST 9.5 (GST 9.5-1 and GST 9.5-2) were cloned by RT-PCR. The deduced amino acid sequences of GST 9.5-1 and GST 9.5-2 clones were identical to those of mGSTA1 and mGSTA2, respectively. Both these subunits were expressed in Escherichia coli to determine the relationships between recombinant mGSTA1-1 and mGSTA2-2 and corresponding subunits of tissue-isolated GST 9.5. The pI values of recombinant mGSTA1-1 and mGSTA2-2 (9.49 and 9.45, respectively) were similar to that of the tissue-isolated isoenzyme (pI 9.5). The reverse-phase HPLC elution profiles and immunological cross-reactivities of recombinant mGSTA1-1 and mGSTA2-2 were also similar to those of the corresponding subunits of tissue-isolated GST 9.5. The catalytic efficiency of recombinant mGSTA1-1 toward (+)-anti-BPDE, 131 mM-1.s-1, was approximately 9.5-to 655-fold higher compared with tissue-isolated mGSTP1-1, mGSTA3-3, mGSTM1-1, and mGSTA4-4. Moreover, the catalytic efficiency of mGSTA1-1 toward (+)-anti-BPDE was about 3.3-fold higher compared with recombinant mGSTA2-2. The mGSTA1 and/or mGSTA2 subunits were expressed to varying degrees in female A/J mouse tissues. For example, mGSTA1, but not mGSTA2, subunit expression was observed in the skin, which is a target organ for benzo(a)pyrene (BP)-induced cancer in mice. On the other hand, the expression of either mGSTA1 or mGSTA2 subunit could not be detected in the lung, which is another target organ for BP-induced cancer in mice. Interestingly, relatively large amounts of both mGSTA1 and mGSTA2 subunits were detected in the kidney. In conclusion, the results of the present study clearly indicate that the A1-type subunit of GST 9.5 is responsible for its exceptional catalytic efficiency in the GSH conjugation of (+)-anti-BPDE, which is the ultimate carcinogen of widespread environmental pollutant BP.