Rat liver glutathione S-transferases consist of binary combinations of three major classes of subunits designated as Ya (Mr = 25,600), Yb (Mr = 27,000) and Yc (Mr = 28,000). We have determined the nucleotide sequences of a cDNA insert in pGTR262 containing partial sequence of a Yc subunit. Sequence comparison with a Ya subunit cDNA clone pGTR261 revealed 70% nucleotide sequence homology and 65% amino acid sequence homology in the overlapping coding regions. Approximately 65% of the amino acid substitutions between these two subunits occur in clusters of two to eight residues. The 3' noncoding sequences of these two subunit cDNA clones are highly divergent in length and in sequences. The 3' noncoding region of pGTR262 cDNA contains open reading frames of 23, 39, and 13 amino acids which are in phase with the deduced Yc subunit sequences but interrupted by termination codons. By RNA blot hybridization analysis, we found that this Yc subunit sequence expression is tissue specific. It is expressed at a reduced level in kidney and testis as compared with liver and hardly at all in heart, lung, seminal vesicles, and spleen. We suggest that the mechanism for the tissue-specific expression of the Yc subunit of rat liver glutathione S-transferases may occur at or prior to the level of RNA processing. From the divergent DNA sequences between the Ya and Yc subunits reported in this communication and their differential induction upon acute phenobarbital administration reported by Pickett et al. (Pickett, C.B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543) we propose that the Ya and Yc subunits of rat liver glutathione S-transferases are encoded by separate gene families or transcriptional units.