Hypersecretion of human GH (hGH) or PRL by human pituitary adenomas is not under normal homeostatic control despite normal receptor function mediating the regulatory effects of hypothalamic peptides for these trophic hormones. This implies that the defects underlying hormonal hypersecretion may not reside at the plasma membrane of the adenoma cell; instead, dysregulation may reside at the hormone gene level. To investigate this, genomic DNA derived from a prolactinoma and a hGH-secreting adenoma were digested with the restriction endonuclease EcoRI and the methylation sensitive restriction endonuclease HpaII and hybridized with the 32P-labeled genomic hGH (2.6 kilobase) probe. Our data revealed hypomethylation of genes of the hGH family (hGH and chorionic somatomammotropin) in the absence of gross abnormalities such as gene translocation. In a similar analysis using a 32P-labeled probe consisting of the EcoRI-BamHI (500 base pair) fragment in the 5'-flanking region upstream of the first exon of the hGH gene, hypomethylation of this specific site of the hGH genes was observed. These results are consistent with the concept that hypomethylation of genes is involved in gene expression. At the same time, protooncogene abnormalities in these adenomas were investigated to delineate any genetic basis for their neoplastic growth. Genomic DNA of adenomas were subjected to Southern blotting analysis using a panel of protooncogene probes. Amplification of the v-fos gene was observed in one prolactinoma. The significance of this observation is discussed.