Previous studies have shown that the hypothalamic concentration of pro-opiomelanocortin (POMC) mRNA and several POMC-derived peptides increases in the rat 4 weeks after orchiectomy and that this increase can be prevented by testosterone replacement. In this study, we have examined the short-term effects of orchiectomy on POMC gene expression in the medial basal hypothalamus (MBH). Adult male rats were studied at various time points between 1 day and 4 weeks after orchiectomy and compared to sham-orchiectomized rats. The MBH was homogenized and, after an aliquot was removed for β-endorphin (β-EP) radioimmunoassay, the RNA was isolated and the amount of POMC mRNA was measured using a solution hybridization SI nuclease protection assay. In the first experiment, POMC mRNA was significantly higher 4 weeks after orchiectomy compared to that of the intact controls: 1.34 ± 0.14vs.0.86 ± 0.04 pg/µg RNA (p < 0.01). Three days after orchiectomy, POMC mRNA was somewhat lower, 0.71 ± 0.06 pg/µg RNA, but not significantly different from the controls. In a second experiment, POMC mRNA levels were measured 1, 2, 3, 4 and 7 days after orchiectomy. At 1 and 2 days after orchiectomy, POMC mRNA was lower than the controls: 0.51 ± 0.02 and 0.52 ± 0.06 vs. 0.70 ± 0.09 pg/µg RNA. Levels then steadily increased to 0.61 ± 0.04, 0.70 ± 0.09 and 0.78 ± 0.11 pg/µg RNA at 3, 4 and 7 days after orchiectomy, respectively. The mean level at 1–2 days after orchiectomy was significantly less than the controls (p < 0.05). In a third experiment, POMC mRNA was again significantly lower 2 days after orchiectomy, 0.74 ± 0.05 pg/µg RNA, compared to 2 days after sham orchiectomy, 1.01 ± 0.12 pg/µg (p < 0.05). The decrease in POMC mRNA that we measured 2 days after orchiectomy, however, was not prevented by our regimen of testosterone replacement. The only significant change in β-EP content was the increase noted 4 weeks after orchiectomy which paralleled the increase in POMC mRNA. Thus, the effects of castration on POMC gene expression in the male rat appear to be biphasic with an initial decrease followed by a much later increase in POMC mRNA levels. The initial fall in POMC mRNA occurs at the time when luteinizing hormone (LH) levels are increasing and may thus play a role in modulating the early postcastration changes in gonadotropin-releasing hormone and LH secretion.