Thirty-four human pituitary adenomas were examined for the presence of Pit-1 mRNA via Northern analysis. All tumors that were immunoreactive with either human (h) PRL or hGH antiserum contained Pit-1 mRNA (8 of 8) as did a small percentage of alpha-subunit only (1 of 3) and null tumors (1 of 9). Two tumors that contained TSH beta mRNA by RNAse protection assay also contained Pit-1 mRNA. Tumors that were immunoreactive with antiserum for hACTH, hFSH beta, or hLH beta without containing PRL, GH, or TSH beta were uniformly Pit-1 negative (0 of 14). Although RNA from the majority of Pit-1-positive tumors (10 of 11) contained primarily a 2.4-kilobase (kb) Pit-1 mRNA transcript, 1 tumor appeared to contain a 1.6-kb mRNA. Analysis of human pituitary autopsy specimens revealed no 1.6-kb Pit-1 mRNA, indicating that this variant may be tumor specific. The wide variety of serum PRL, GH, and insulin-like growth factor-I levels in patients with Pit-1-positive tumors suggests that although Pit-1 is necessary for PRL and GH production, it is not uniformly associated with hormone production, as determined by either immunohistochemistry or elevated serum levels. The expression of Pit-1 mRNA in a small fraction of null and alpha-subunit only tumors indicates that there may be a previously unsuspected diversity of origin within these particular subsets of adenomas.