Alpha-melanocyte-stimulating hormone (α-MSH) stimulates prolactin (PRL) release and mammotrope proliferation in mouse anterior pituitary glands. The present study investigated the regulation of melanocortin-3 receptor (MC3-R) mRNA levels in mice using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Relative MC3-R mRNA levels in the anterior lobes of female mice increased between 20 and 45 days of age, and a significant difference in MC3-R mRNA expression between sexes was seen at 45 days. Ovariectomy decreased MC3-R mRNA expression in the female anterior lobes, and estradiol-17β (E<sub>2</sub>) treatment increased MC3-R mRNA levels in ovariectomized mouse anterior lobes and cultured anterior pituitary cells. E<sub>2</sub> treatment increased proopiomelanocortin (POMC) mRNA levels in ovariectomized mouse neurointermediate lobes and cultured neurointermediate pituitary cells. On the other hand, E<sub>2</sub> treatment did not affect POMC mRNA expression in mouse anterior lobes or cultured anterior pituitary cells. These results suggest that α-MSH directly stimulates PRL release and mammotrope proliferation through MC3-Rs expressed in mammotropes, while estrogen stimulates MC3-R gene transcription in the anterior pituitary cells and POMC gene transcription in the intermediate lobes. In lactating mice, POMC mRNA levels in the neurointermediate lobes were elevated compared with in non-lactating mice. The present study suggests that α-MSH is involved in augmented PRL secretion by estrogen and during lactation.