Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with diverse effects, was originally isolated as a hypothalamo-hypophyseal peptide. Subsequent studies showed highest levels of PACAP in the testis after the brain, suggesting that it influences the development and functioning of spermatozoa. Indeed, it has been proven that PACAP has an effect on spermatogenesis, both locally and via influencing the hypothalamo-hypophyseal-gonadal axis. We have previously described that PACAP increases human sperm motility. The aim of the present study was to investigate spermatogenesis in PACAP gene deficient mice. Sperm head and testis morphology, motility and expression of spermatogenic factors were studied in mice lacking endogenous PACAP and their wild type littermates. Routine light microscopy revealed no marked difference in the structure of testis and epididymis between PACAP KO and wild type mice. Investigating sperm head morphology in PACAP deficient mice revealed that both the longitudinal and transverse diameters were significantly lower in PACAP deficient mice. The shape of the head was not markedly different, as shown by scanning electron microscopy. Western blot, PCR and immunohistochemical investigation showed that the expression of the transcription factor Sox9, which plays an important role in spermatogenesis, was markedly lower in PACAP KO mice. In summary, these results show that there are differences in both the sperm morphology and the expression of transcription factors important in spermatogenesis in PACAP gene deficient mice. These differences may account for the lower motility and the decreased fertility in these mice and indicate the importance of PACAP in reproductive processes.