To elucidate whether testosterone (T) replacement to castrated male rats may promote luteinizing hormone-releasing hormone (LH-RH) biosynthesis in the hypothalamus, an RNA-blot hybridization assay was employed. Poly(A) mRNA fractions from hypothalami (40-50) of intact, castrated plus vehicle, and castrated plus T group were isolated, blotted on nitrocellulose paper and hybridized with 32P-end labelled LH-RH oligonucleotides (29 mer) which is complementary to the rat LH-RH mRNA. LH-RH-like mRNA level markedly attenuated 2 weeks following castration and T replacement significantly increased LH-RH-like mRNA level, comparable to about 80% of that observed in intact male rats. In addition, LH-RH contents and release from hypothalami were studied, to examine the correlation with LH-RH gene expression and secretory activity of LH-RH. Castration significantly reduced LH-RH contents and LH-RH release in vitro. T replacement restored LH-RH contents and release comparable to those shown in the intact group. This study demonstrates for the first time that T replacement increases LH-RH-like mRNA level in the hypothalami of castrated rats suggesting that T may act at the pretranslational level. This change in LH-RH gene expression is parallel with alteration of LH-RH content and release.