Suppressin (SPN) is a novel polypeptide that is synthesized and secreted by normal rat pituitary cells and a rat pituitary tumor cell line, GH3. Specifically, SPN is a negative regulator of growth that inhibits lymphoid and neuroendocrine cell proliferation. The objective of the present study was to identify the cells in the normal rat pituitary that produce SPN. A double immunofluorescence technique, using antibodies to SPN in conjunction with antibodies to the six major adenohypophyseal hormones, was used to colocalize SPN and a specific hormone in a single dispersed pituitary cell. The results of these experiments showed that, on the average, 42% of the cells in the pituitary produce SPN. Suppressin production in the pituitary was restricted to the adenohypophysis. The SPN-producing population in the pituitary was composed of somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs, while gonadotrophs did not produce SPN. Additionally, a PRL reverse hemolytic plaque assay was used to examine SPN production in lactotrophs. The results of these experiments showed that SPN production and the amount of PRL secreted covaried. Specifically, SPN production was observed primarily in non-PRL-secreting lactotrophs or in lactotrophs secreting a high amount of PRL. The results of these experiments suggest a potential regulatory relationship between the synthesis and secretion of SPN and PRL. In summary, this report provides the first identification of SPN-producing cells in the pituitary and shows that SPN production occurs primarily in somatotrophs and lactotrophs.