Neuropeptide Y (NPY) peptide concentrations in the arcuate nucleus have recently been shown to be modulated by gonadal steroids in the male rat. The present study was designed to determine whether NPY messenger RNA (mRNA)-synthesizing cells in the arcuate nucleus (Arc) of the male rat are regulated by testosterone (T) and whether there is a sexual dimorphism in the expression of the NPY gene in this region. In situ hybridization and quantitative autoradiography were used to assess the level of NPY gene expression in the Arc. In the first experiment, NPY mRNA levels were measured in the Arc of intact, castrated, and castrated male rats treated with T to maintain physiological (1.3 +/- 0.1 ng/ml) and supraphysiological (5.3 +/- 0.4 ng/ml) plasma levels of T. A 2-week castration produced a modest but significant decrease in NPY mRNA levels in the Arc (P < 0.05). Replacement with either physiological or supraphysiological levels of T prevented the effect of castration on NPY gene expression, and there was no further potentiation of NPY gene expression in those animals that received high levels of T. In the second experiment, NPY gene expression was compared throughout the Arc between intact male and female rats at 1800 h on the afternoon of proestrus. Comparison of NPY gene expression throughout the rostro-caudal extent of the Arc showed that male rats had significantly more NPY mRNA-containing cells than female rats (P < 0.01). This difference was most strikingly observed in the caudal portions of the nucleus (3.80 mm caudal to bregma). No difference was detected in the mean levels of NPY gene expression in the Arc between male and female rats. These data demonstrate that 1) NPY gene expression throughout the arcuate nucleus is modulated by T in male rats, and 2) a marked regional sex difference exists in the distribution of NPY mRNA-containing cells in the caudal extremity of the Arc. It is hypothesized that gonadal hormones may exert both organizational and activational effects upon NPY neurons in the Arc.