Results of studies designed to investigate the origin of the diversity of glutathione S-transferase (GST) isozymes in human tissues indicated that human muscle has at least three forms of GST with pI values of 5.0, 5.1, and 5.2 that are distinct from GST isozymes characterized so far. The major muscle isozyme which was expressed in all the six samples analyzed in this study was a unique GST of pI 5.2 that was designated as GST zeta. It had a blocked N-terminal and did not correspond to any of the known three classes (alpha, mu, or pi) of human GST as evidenced by its immunological properties and substrate specificities. The N-terminal regions of human muscle GST 5.1 and 5.0 had identical amino acid sequences except at residue 5, but demonstrated significant differences in amino acid composition and substrate specificities. These two isozymes showed homology with the mu class of human GST in their N-terminal region and were also immunologically related to the mu class of human GST although their subunit molecular weight values (Mr 23,000) were lower than that reported for GST psi. The substrate specificities of these isozymes were also significantly different from those of other human GST isozymes characterized so far. Significantly, muscle tissue did not express the alpha class of GST isozymes; however, two other isozymes were identified, GST 4.8 and GST 4.5, which had identical N-terminal amino acid sequences that were similar to that reported for the pi class of human GST. GST 4.8 was present in all six samples analyzed in this study whereas GST 4.5 was present in only two of these samples, indicating a possibility of polymorphism at the GST3 locus. This study indicated the occurrence of at least three distinct isozymes in muscle tissue, providing further evidence for tissue specific expression of GST isozymes in humans.