We have recently characterized two members of a novel family of murine testis specific serine kinases, tssk-1 and tssk-2, expressed exclusively in spermatids undergoing spermiogenesis. Using a differential screening approach we have isolated a third family member, tssk-3. The open reading frame of tssk-3 encodes a protein of 275 amino acids, consisting essentially of a serine/threonine protein kinase domain only. In contrast, tssk-1 and -2 have distinct, approximately 100 amino acid domains located C-terminally to the kinase domain. Immunoprecipitation experiments revealed that while tssk-1 and tssk-2 form detergent resistant complexes, tssk-3 is not associated with either protein. Expression of tssk-3 was induced at puberty, persisted during adulthood and was restricted to the interstitial Leydig cells of post-pubertal males.