Expression of P3H2 (Leprel1) and P3H3 (Leprel2) but not P3H1 (Leprecan) is down-regulated in breast cancer by aberrant CpG methylation in the 5' regulatory sequences of each gene. Methylation of P3H2 appears specific to breast cancer as no methylation was detected in a range of cell lines from other epithelial cancers or from primary brain tumours or malignant melanoma. Methylation in P3H2, but not P3H3, was strongly associated with oestrogen-receptor-positive breast cancers, whereas methylation in P3H3 was associated with higher tumour grade and Nottingham Prognostic Index. Ectopic expression of P3H2 and P3H3 in cell lines with silencing of the endogenous gene results in suppression of colony growth. This is the first demonstration of epigenetic inactivation of prolyl hydroxylases in human cancer, implying that this gene family represents a novel class of tumour suppressors. The restriction of silencing in P3H2 to breast carcinomas, and its association with oestrogen-receptor-positive cases, suggests that P3H2 may be a breast-cancer-specific tumour suppressor.