Expression of the human parathyroid hormone (PTH)/ PTH-related peptide receptor (PTHR) gene is controlled by three promoters, P1-P3, P1 functions specifically in kidney, whereas P2 is ubiquitously active. P3 is also widely active, although more so in kidney than other tissues. However, only P2 functions at midgestation. We examined the role of methylation in controlling PTHR promoter activity. Function of all promoters was inhibited by CpG methylation in vitro. Significantly, P1 is selectively hypomethylated in adult kidney in vivo strongly suggesting that demethylation is required for renal P1 function. Moreover, this pattern is established by 11.75 weeks of fetal age, several weeks prior to the onset P1 activity. P3 is unmethylated at midgestation, although it is inactive at this stage of development, and thus exhibits characteristics of both tissue-specific and ubiquitously active promoters. These results show that adult methylation patterns of P1 and P3 are established several weeks prior to their induction, indicating that their function requires factors expressed late in development.