Forty-four human growth hormone (GH)-producing adenomas were investigated immunohistochemically for the concomitant localization of GH, prolactin (PRL), and glycoprotein hormone alpha subunit. Immunoreactivity for GH, PRL, and the alpha subunit was found in 44, 37, and 36 cases, respectively. By double immunohistochemical staining, 24 of 27 cases showed colocalization of GH and alpha subunit, with the numbers of tumor cells showing double staining varying from case to case. The colocalization of GH and alpha subunit was noted in some normal pituitary cells. In adenoma cells, the colocalization of GH and PRL, and PRL and alpha subunit, was observed in 9 and 12 cases, respectively. The normal pituitary gland showed only occasional colocalization of GH and PRL, or PRL and alpha subunit. We found that GH-producing adenomas are plurihormonal at the individual tumor cell level, with coexpression of GH-alpha subunit, GH-PRL, and PRL-alpha subunit. The colocalization of GH and alpha subunit may be an expression of a subpopulation of normal anterior pituitary cells (with GH-alpha subunit coexpression), but the more frequent coexpression of GH-PRL may be pathological, accompanying tumorigenesis of the anterior pituitary cells, in which a pituitary-specific transcriptional factor, pit-1, may play a role.