We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.