Glutathione S-transferases (GSTs) are dimeric proteins that come from a multigene family. They can be grouped into five classes (alpha, mu, pi, sigma, theta) based on the degree of amino acid homology of their subunits. These GST isozymes are able to catalyze the conjugation of glutathione with a wide variety of electrophiles, thereby protecting important cellular constituents from electrophilic attack. In the present study, the distribution of the Ya and Yc subunits from the alpha family, as well as the Yb1 and Yb2 subunits from the mu gene family was examined using immunocytochemistry in the adult rat testis and epididymis. The results of these four GST subunits were also compared with two other subunits, the Yf and Yo proteins, which have already been investigated in our laboratory [Veri et al. (1993), J. Androl., 14:23-44; Veri et al. (in press), J. Androl.]. In the testis, Leydig cells were intensely stained for all six subunits. Within the seminiferous epithelium, Sertoli cells were reactive only for antibodies raised against the Ya, Yb1 and Yf subunits. Among germ cells, all spermatogonia, spermatocytes and step 1-15 spermatids were virtually unreactive for each of the six GSTs. However, moderate to intense staining was seen over steps 16-19 spermatids with the anti-Yo and anti-Ya antibodies, and intense staining over step 19 spermatids with the anti-Yb1 and anti-Yb2 antibodies. In the rete testis, Yf, Yo, Yb1, and Yb2 subunits were intensely reactive over the epithelial cells with weak staining for Yc and no staining for Ya antibodies. Interestingly, in the efferent ducts the Yc, Yb1, and Yf proteins were intensely reactive over ciliated cells, whereas only the Yc protein was intensely reactive over nonciliated cells. In the epididymis, immunoreactivity varied among the principal and basal cells of a given epididymal region for each GST antibody. In the case of principal cells, several of the GSTs showed a similar immunostaining pattern along the tubule. Although not identical in intensity of reaction, the Yc, Yb1, Ya and Yo proteins showed an increase in staining intensity from the proximal to distal segments of the epididymis. In contrast, the Yb2 protein was intensely expressed only in the distal caput with weak levels throughout the rest of the epididymis. The Yf reactivity was strongest from the distal initial segment to the distal caput and unreactive in the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)