The cytosolic Mu-class glutathione S-transferases (GST) present in human liver, skeletal muscle and testis comprise three distinct neutral-type subunits (N₁ N₂ and N₃) which hybridize to form homodimers or heterodimers. The two N₁-type subunits, which represent allelic variants encoded by the hepatic Mu-class GST locus, are designated N_1^a and N_1^b. Three isoenzymes containing N₁-type subunits have been purified from one liver specimen by sequential affinity chromatography, hydroxyapatite chromatography and chromatofocusing. The skeletal muscle GST N_1^aN₂, N_1^bN₂, N₂N₂ and N₂N₃ were purified by a combination of affinity chromatography and anion-exchange f.p.l.c. followed by either chromatofocusing f.p.l.c. or hydroxyapatite h.p.l.c. In muscle the expression of the N₁-type subunit, but not of the N₂ and N₃ subunits, was found to differ from specimen to specimen. The N₁-type subunits were absent from about 50&37 of samples analyzed, and the purification results from four different specimens are presented to illustrate the phenotypic variation of skeletal muscle GST. The isolation of a homodimeric Mu-class isoenzyme, called GST N₃N₃, from human testicular tissue was achieved in two chromatographic steps; namely affinity chromatography followed by anion-exchange f.p.l.c. The neutral-type GST subunits have been defined by the decreasing isoelectric points of the homodimeric enzymes; GST N_1^aN_1^a, N_1^bN_1^b, N₂N₂ and N₃N₃ have pl values of 6.1, 5.5, 5.3 and 5.0. SDS/PAGE showed that N₁, N₂ and N₃ have M_r values of 26700, 26000 and 263000 respectively. The N₁, N₂ and N₃ subunits are catalytically distinct, with the N₁-type subunits possessing high activity for trans-4-phenyl-3-buten-2-one and N₂ having high activity towards 1,2-dichloro-4-nitrobenzene. The N-type subunits may also be distinguished immunochemically; antisera raised against the testicular GST N₃N₃ cross-reacted with the N₃ subunit, and showed no reactivity towards either the N₁-type or the N₂ subunit. N-terminal amino acid sequence analysis supported the electrophoretic evidence that the N₂ subunit in GST N_1^aN₂, N_1^bN₂, N₂N₂ and N₂N₃ represents the same polypeptide. The peptides obtained from CNBr digests of N₂ were subjected separately to automated amino acid sequencing, and the results indicate that N₂ is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH₄ [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8542-8554]. GST N₂N₂ is probably identical with GST4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST4 there is complete identity between the two enzymes. The N₃ subunit, which is expressed in skeletal muscle and testis possesses a blocked N-terminus. Automated amino acid sequencing of a CNBr-derived peptide allowed 14 residues of the N₃ subunit to be identified. This data indicated that testicular GST N₃N₃ is likely to be identical to brain/testis μ [Campbell, Takahashi, Abramovitz, Peretz & Listowsky (1990) J Biol. Chem. 265, 9188-9195].