The mRNA expression pattern of dipeptidyl peptidase (DPP) 8 and DPP9, two DPP4 homologues, was studied before and revealed a broad tissue distribution (Abbott et al. 2000; Olsen and Wagtmann 2002; Qi et al. 2003; Ajami et al. 2004). In this study, protein expression and activity of DPP8 and DPP9 were investigated in male reproductive tissues of different mammals. Based on specific DPP activities and inhibition profiles, the proline-selective DPP activity in the bovine and rat testis could predominantly be attributed to DPP8/9 and not to DPP4. This is in contrast to the epididymis where most of the activity was due to DPP4. Bovine sperm preparations had a very low or undetectable DPP8/9 activity. After characterization of polyclonal antibodies specific for DPP8 or DPP9, we could localize both enzymes in seminiferous tubules of the testis. A specific staining for DPP9 was found associated with spermatozoids embedded in the epithelium, just before their release into the lumen, and in spermatids. DPP8 was localized in spermatozoids in an earlier stage of maturation. These findings can help to give an insight in the physiological role of DPP4-like enzymes in the male reproductive system. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.