Specific angiotensin II (Ang II) binding and functional responses have been studied in a number of male and female reproductive structures, but to date have not been characterized in the epididymis. Some epididymal functions, including smooth muscle contraction and regulation of fluid and electrolyte balance, are mediated by Ang II in other tissues. This study demonstrates specific, saturable (63 fmol/mg protein), high affinity (Kd, less than 1 nM) Ang II-binding sites in the epididymis. These binding sites, localized in the circumference of the epididymal tubule and most concentrated within the proximal cauda, are present throughout the caput, corpus, and remaining cauda epididymis. Ang II caused powerful expulsions of spermatozoa in segments of epididymis in situ. These results suggest Ang II involvement in epididymal function.