The expression of the phosphoglycerate mutase locus Pgam-2 which synthesizes the muscle-specific PGAM-B subunit was analyzed in the testis of the mouse. No PGAM-B activity was detected in testes of newborn mice, in which only the PGAM-AA isozyme was observed. PGAM-B was first observed between Day 14 and Day 16 of postnatal development. In adult males approximately 50% of total PGAM activity is contributed by the PGAM-B subunit and 50% by the PGAM-A subunit. Immunohistochemical studies show that in the testis PGAM-B is localized exclusively in germ cells. PGAM-B is detected in pachytene spermatocytes and in spermatids, but not in earlier stages of spermatogenesis. The muscle-specific PGAM isozyme was also found in testes of bull, cat, and rat, as well as in human sperm. PGAM-B might thus be useful as a marker for germ cell differentiation, along with other germ cell-specific proteins.