AbstractThe protein phosphatase 1cγ (PP1cγ) gene is required for spermatogenesis. Males homozygous for a null mutation are sterile, and display both germ cell and Sertoli cell defects. As these two cell types are physically and functionally intimately connected in the testis, the question arises as to whether the primary site of PP1cγ action is in Sertoli cells, germ cells, or both. We generated chimeric males by embryo aggregation to test whether wild type Sertoli cells are capable of rescuing mutant germ cells. To distinguish between the desired XY‐XY chimeras and uninformative XX‐XY chimeras, we designed an adaptation of the single nucleotide primer extension (SNuPE) assay. None of the XY‐XY chimeras sired pups derived from mutant germ cells, indicating that the protein is required in germ cells for production of functional sperm. Analysis of a chimeric testis revealed intermediate phenotypes when compared with PP1cγ−/− testes, suggestive of cell nonautonomous effects. We conclude that PP1cγ is required in a cell autonomous fashion in germ cells. There may be an additional cell nonautonomous role played by this gene in testes, possibly mediated by defective signaling between germ cells and Sertoli cells. Mol. Reprod. Dev. 65: 157–166, 2003. © 2003 Wiley‐Liss, Inc.