The seminiferous tubule of the testis contains a rich variety of microtubule networks and of microtubule-associated proteins (MAPs). Tau is a heat-stable MAP previously believed to be limited in its expression in mammals to the nervous system. We have identified tau in rat and bovine testis, a unique non-neuronal location, using biochemical, molecular, and immunologic approaches. SDS-PAGE of ammonium sulfate-fractionated, testis heat-stable MAPs resulted in an enrichment of bands that comigrated with rat brain tau. Only the 35-45% precipitated ammonium sulfate fraction induced microtubule assembly. Immunoblotting with monoclonal anti-tau antibodies demonstrated tau immunoreactivity in these testis MAP preparations. Northern analysis of total rat testis RNA demonstrated a 1.7-kb band that hybridized with a 51-nucleotide oligomer complementary to a conserved portion of the tau transcript. This 51-mer identified a similar 1.7-kb minor band and an additional 6-kb major band in Northern analysis of total rat brain RNA. Finally, in the bull testis, immunohistochemistry localized tau to the spermatid manchette, a transient, cross-linked microtubule network of unknown function. As spermatid elongation begins, the manchette forms a sheath around the posterior aspect of the nucleus, but, by the completion of nuclear condensation, the manchette is largely disassembled. Tau most likely plays a structural role in the manchette; however, tau immunoreactivity also was observed in late stage I spermatids prior to manchette formation, suggesting that tau may serve a function in manchette assembly.