The pituitary-specific transcriptional factor, Pit-1, is a member of the POU-domain family, which has a role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Recently, specific DNA-dependent interactions have been observed between Pit-1 and nuclear receptors, including: thyroid hormone receptor; retinoic acid receptor; glucocorticoid receptor; and estrogen receptor (ER). The cooperative interaction between Pit-1 and ER required for prolactin enhancer activity in rat pituitaries has been suggested. We analyzed the expression of Pit-1 messenger ribonucleic acid (mRNA) and ER mRNA in 15 human prolactin-producing adenomas using nonradioisotopic in situ hybridization. Their products were also studied by immunohistochemical analysis. Pit-1 mRNA was detected in 12 (80%) of 15 prolactin-producing adenomas. On the other hand, ER mRNA was detected in 14 (94%) of adenomas studied. mRNAs of Pit-1 and ER were detected more frequently than immunohistochemical expression of their products. By combined in situ hybridization and immunohistochemical examination, Pit-1 mRNA and ER mRNA were often colocalized with prolactin immunoreactivities. The colocalizations of Pit-1 mRNA and ER protein were observed in adenoma cells. The high incidence of the expression of ER mRNA in prolactin-producing adenomas may suggest cooperative interactions between Pit-1 and ER in functional differentiation and development of prolactin-producing adenomas.