Previous studies from this laboratory have demonstrated immunostainable beta-endorphin-like material (beta-EP) in Leydig cells and epithelia of the epididymis, seminal vesicle and vas deferens of the rat. These observations would be strengthened if it could be demonstrated that they were not a peculiarity of the rat. Accordingly, we now present immunocytochemical evidence for the presence of beta-EP in the Leydig cells of mouse, hamster, guinea pig and rabbit. No immunoreactive material was identified in Sertoli, myoid, endothelial or germ cells of any of the species examined. Immunostainable beta-EP was also demonstrated in the epididymides of mouse, guinea pig, rabbit, and rat, but not hamster. Immunostainable material was also present in the epithelia of the vas deferens and seminal vesicles of mouse and rat, the only two species thus far examined. Since beta-EP was present in Leydig cells, we wondered whether this peptide could be identified in other steroid-producing tissues. When rat ovaries and adrenals were reacted with anti-beta endorphin, staining was demonstrated in corpus luteum and adrenal cortex. No staining was observed in the adrenal medulla or other portions of the ovary. In order to determine whether the beta-EP detected in the testis and epididymis was derived from a pituitary source, animals were hypophysectomized and tissues examined 2 weeks later. Both the Leydig cells and the epididymal epithelium remained immunostainable. In summary, immunostainable beta-EP has been identified in Leydig cells of five species. Stainable material is also present in the epithelium of other portions of the male reproductive tract and in steroid-secreting cells of the ovary and the adrenal. Such beta-EP may have a paracrine function in the testis and other portions of the male reproductive tract.