Excessive production of a parathyroid hormone-related protein (PTHrP) by tumours commonly results in the syndrome of humoral hypercalcaemia of malignancy. We have investigated whether epigenetic changes play a role in over-expression of the PTHrP gene, using cultures lung cells as a model system. Study of the methylation status of CpG dinucleotides in the 5' region of the gene showed that in normal cells the CpG island was completely unmethylated. In the lung squamous cell carcinoma cell line, BEN, two-thirds of the CpG island was substantially methylated. RT-PCR analysis showed that this heavy methylation did not prevent expression of any of the three PTHrP gene promoters. This is a surprising finding, since methylation is usually associated with inhibition of gene activity. Methylation of the 5' non-coding region of the PTHrP gene may not play a role in the regulation of adjacent promoters. Alternatively, maintenance of a demethylated state in the 170 bp at the 3' end of the CpG island may be fundamental for the use of PTHrP promoters.