Tumor production of a parathyroid hormone-related peptide (PTHrP) is a common cause of the syndrome of humoral hypercalcemia of malignancy, which is frequently associated with renal cell carcinomas. Why certain renal cell carcinomas produce PTHrP while others do not is unknown. Using a system of 12 human renal carcinoma cell lines which either do (n = 6) or do not (n = 6) produce PTHrP, we found that the expression of the PTHrP gene in these cell lines is controlled at the transcriptional level. Transfection studies failed to demonstrate variation in PTHrP promoter activity in these cell lines sufficient to account for the differential PTHrP expression, implicating a cis-acting mechanism. Transcription of the PTHrP gene in these cell lines was found to correlate with the methylation state of specific CpG dinucleotides located within the promoter region but outside a CpG island. The functional importance of this mechanism of control was confirmed by the ability of the demethylating agent, 5-azacytidine, to induce PTHrP mRNA expression in previously nonexpressing cell lines.