Estrogen receptor-alpha (ER-α) is important for male reproduction in mammals; however, no information is available on ER-α protein distribution in the testes of fishes. The cellular localization of the rainbow trout (Oncorhynchus mykiss) ER-α (rtER-α) protein, throughout the annual reproductive cycle was determined in this study. An antibody was designed based on a 15-amino acid sequence from the D-domain of the rtER-α, and its specificity was confirmed using Western blot analysis. Immunohistochemical analysis revealed rtER-α protein to be present only in the testicular interstitium, at every stage of the annual reproductive cycle. The localization of rtER-α protein in the interstitial fibroblasts, the Leydig cell precursor in the rainbow trout, suggests a role for estrogens in the differentiation of these precursor cells into mature Leydig cells. This is the first study to report the cellular localization of an estrogen receptor protein in the testis of any fish species.