A rat testicular Mu-class glutathione S-transferase (GST) resolved by reversed-phase high performance liquid chromatography cross-reacted with peptide sequence-specific antisera raised against the human hGSTM3 subunit. Electrospray ionization mass spectrometry indicated that this rat GST subunit (designated rGSTM5 in this report) has a significantly greater molecular mass (26,541 Da) than the other rat GST subunits. The mouse homologue (mGSTM5 subunit) was also identified and characterized by high performance liquid chromatography and electrospray ionization mass spectrometry. Sequence analysis of rGSTM5 peptide fragments and the sequence deduced from a cDNA clone showed that the protein is highly homologous to the hGSTM3 and murine mGSTM5 subunits. All three GSTs of this subclass have N- and C-terminal extensions with C-terminal cysteine residues, but the two penultimate amino acids near the C terminus are divergent in the three species. The proteins of this class Mu subfamily have similar catalytic specificities and mechanisms, are all cysteine rich, are found mainly in testis, and share characteristics that distinguish them from other GSTs. Moreover, the rGSTM5 subunit isolated from rat testis was not found in heterodimeric combination with other common Mu-class GST subunits. As the rGSTM5, mGSTM5, and hGSTM3 subunits are structurally more closely related to each other than they are to other Mu GSTs, it is proposed that they be considered a functionally distinct and separate subfamily within class Mu. The identification of this unique mammalian GST subclass could advance strategies for interspecies comparisons of GSTs and provides a rodent model for studies on functions and regulatory mechanisms for human GSTs.