Metallothionein-3 (MT-3) is a new MT gene-family member that inhibits survival of rat neurons cultured in presence of brain extracts. Contrary to other MT genes, which are expressed in most tissues and which are highly inducible by metals, MT-3 expression was reported to be mainly in the brain, and it failed to respond to metals in vivo. We show here that MT-3 mRNA is present in several organs other than the brain, as assayed by Northern analyses. In the rat, MT-3 mRNA was detected in the testis, prostate, epididymis, tongue, ovary, uterus, stomach, heart, and seminal vesicles. The MT-3 mRNA levels in the testis, epididymis, prostate, and tongue were 22% of those in brain, while in ovary, uterus, and stomach, they were 4% of the brain level, and they were lower still in the other organs. The MT-3 gene was not inducible by CdCl2 or lipopolysaccharide in rat testis and prostate. In the mouse and the human, relative MT-3 mRNA levels were lower than those found in the rat when compared with those present in brain. Testicular MT-3 transcript levels remained quite constant during rat postnatal development in animals aged from 6 to 43 days. In situ hybridization analyses on human testis sections showed that MT-3 mRNA was present at different levels in both the Leydig cells and the seminiferous tubules. In orchiectomized rats, prostatic MT-3 mRNA was decreased by 75%, and injections of dihydrotestosterone restored MT-3 mRNA levels to control values. Overall, these results show that MT-3 tissue-specific gene expression is broader than previously reported and provide new experimental systems to study the function and mechanism of action of the MT-3 protein.