During spermiogenesis in the guinea pig, the spermatid plasma membrane becomes sequentially segregated into three domains of distinct composition. We have previously shown that plasma membrane proteins appear on the cell surface in a temporally regulated manner such that proteins localized to the same domain reach the surface membrane at the same time in sperm development. Fertilin is a cell surface protein restricted to the whole head of testicular sperm; like other proteins restricted to this membrane domain, it does not appear on the cell surface until late (steps 11-13) in spermiogenesis. Using confocal microscopy of immunofluorescently labeled testicular sections, we demonstrate that the pre-beta subunit of fertilin is present in pachytene spermatocytes. It is initially observed in long, strand-like structures that likely represent the endoplasmic reticulum; it later appears in a punctate distribution in the cytoplasm of early spermatids prior to its appearance on the surface membrane in late elongating spermatids. Immunoblotting experiments confirm the presence of the fertilin pre-beta subunit in spermatocytes and early spermatids at the same apparent molecular weight as in later stages. These results suggest that the appearance of fertilin pre-beta subunit on the spermatid surface is regulated by a post-translational mechanism.