Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-α-expressing cells and the physiological roles of TGF-α within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-α mRNA-expressing cells, and to clarify the involvement of TGF-α in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-α mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-α mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-α mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17β (E2) and TGF-α treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-α treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-α with anti-TGF-α-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-α mRNA expression in ovariectomized female mice revealed that E2 increases TGF-α mRNA levels. These results indicate that the TGF-α produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner.