Rat spermatozoa were unusually rich in a-glycerophosphate dehydrogenase (GPD) activity. Significant amounts appeared in the testes coincident with the first appearance of mature spermatozoa, and the increased enzyme activity of the testes paralleled the increasing numbers of step 18-19 spermatids present during maturation. The GPD activity of the epididymis was also due to the spermatozoa contained therein. When spermatogenesis was prevented by making an immature testis cryptorchid, GPD activity failed to develop in the testis or associated epididymis. When the germ cells of a mature testis were destroyed by administering dimethylbenzanthracene to the rat, no spermatozoa or GPD activity were present 38 days later; however, the epididymis still retained spermatozoa and GPD activity. Estrogen administered to an adult male rat markedly decreased the spermatozoa content and the GPD activity in both the testis and epididymis; estrogen also prevented the normal development of spermatogenesis and GPD in the maturing rat testis. Testicular GPD activity was not affected by the thyroid hormone, androgen, or gonadotropins. Other species had variable amounts of testicular and spermatozoan GPD activity, but human, dog and horse testes and spermatozoa were practically devoid of this enzyme. (Endocrinology76: 1055, 1965)