Cytosolic glutathione S-transferases (GSTs) from rat ovaries and testis were purified by a combination of GSH and S-hexylglutathione affinity chromatography. The isolated GSTs were subjected to reverse-phase HPLC, electrospray MS and N-terminal peptide sequencing analysis. The major GST isoenzymes expressed in ovaries are subunits A3, A4, M1, M2 and P1. Other isoenzymes detected are subunits A1, M3 and M6*. In rat testis, the major GST isoenzymes expressed are subunits A3, M1, M2, M3, M5* and M6*. Subunits A1, A4 and P1 are expressed in lesser amounts. We could not detect post-translational modifications of any GSTs with known cDNA sequence. The molecular masses of subunits M5* and M6*, two class-Mu GSTs that have not been cloned, were determined to be 25495 and 26538 Da respectively. An N-terminally modified protein from rat testis with molecular mass 25737 Da was isolated from the S-hexylglutathione column. Results from internal peptide sequencing analysis indicate that this is a novel class-Alpha GST that has not been previously reported. We designate this protein rGSTA6*.