β-Endorphin (β-END) is an endogenous opioid peptide derived from the common precursor proopiomelanocortin, together with adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). Although the roles of ACTH and MSH in fish are well known, the roles of circulating β-END have not been elucidated. In the present study, we evaluated the biological roles of β-END in the goldfish. First, we cloned the cDNAs of the delta opioid receptor (DOR), kappa opioid receptor (KOR), and mu opioid receptor (MOR) from the brain of the goldfish. Second, we analyzed the tissues that expressed these genes by using reverse transcription polymerase chain reaction. Among the several tissues that contained the opioid gene transcripts, the mRNAs of DOR, KOR, and MOR were detected in interrenal cells of the head kidney, which produce cortisol. On the basis of these results, the effects of β-END on cortisol release were examined in vitro. β-END alone suppressed the basal release of cortisol in a dose-dependent manner. Moreover, β-END inhibited the cortisol-releasing activity of ACTH 1–24 . Therefore, it is probable that the role of β-END in the interrenal cells is the suppression of cortisol release. Interestingly, the suppression of cortisol release was not observed with N-acetyl-β-END, indicating that acetylation decreases the activity of β-END in interrenal cells.