We have previously shown that a major group of 28-30 kDa proteins decreases after the LH surge in bovine granulosa cells (GC). In the present study, we have characterized two proteins in this group in search of factors that may intervene in folliculogenesis and oocyte maturation. Polyclonal antibodies raised against 28 kDa or 29 kDa bovine GC proteins were used to screen a complementary DNA (cDNA) expression library. This resulted in the characterization of two isoenzyme subunits for alpha class glutathione S-transferase, named bGSTA1 and bGSTA2. Both bGSTA1 (25.4 kDa, pI 8.9; 791 bp cDNA; GenBank Accession No. BTU49179) and bGSTA2 (25.6 kDa, pI 7.2; 959 bp cDNA; GenBank Accession No. AF027386) have 222 amino acids. The deduced amino acid sequences were compared and showed 82% (bGSTA1) and 74% (bGSTA2) identity to human GSTA1, whereas bGSTA1 and bGSTA2 are 81% identical to each other. The bGSTA2 represents a novel GSTA subunit because it harbors a specific 16 amino acid sequence not found in any other species and GST classes. Northern blots showed that bGSTA1 and bGSTA2 are coexpressed and are tissue specific with single transcripts of 1.2 kb and 1.4 kb, respectively for bGSTA1 and bGSTA2. The messenger RNA (mRNA) were detected in GC, corpus luteum, adrenal gland, testis, liver, lung, thyroid, kidney and cotyledon, and the relative abundance of their mRNA varied. Ratios of bGSTA1/bGSTA2 mRNA vary between tisssues, indicating that expression of these genes is controlled differently. Immunohistochemistry observations revealed that expression of GSTA is cell specific, being associated with GC and theca cells, small luteal cells, Leydig cells, hepatocytes, adrenal cortex, specific chromaffin cells in the adrenal medulla, renal proximal convoluted tubular cells, and Clara cells in the bronchioles. Studies in vivo showed that levels of mRNA for bGSTA1 were elevated in follicular wall of preovulatory follicles before hCG treatment, but decreased by 77% 12 h after hCG injection. However, in FSH stimulated preovulatory follicles, the decrease in mRNA for both GSTAs was only 21% at 24 h following hCG injection. We concluded that bGSTA1 and bGSTA2 expression is tissue- and cell-specific, is associated with steroidogenically active cells, and is hormonally regulated by gonadotropins in the bovine ovarian follicle.