The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis.