Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility.