Recent experiments in cultured bovine adrenocortical cells show that the previously observed phenotypic switching of CYP17 (steroid 17 alpha-hydroxylase) expression is preceded at a much earlier time by changes in methylation in the CYP17 5' flanking region. Two CpG sites that are methylated in the adrenal cortex in vivo were observed to undergo rapid demethylation when adrenocortical cells were placed in culture. Two adjacent CpG sites that are also methylated in vivo did not demethylate; these two sites are completely nonmethylated in fibroblasts. All CpG sites downstream, in the promoter or coding region, are always methylated in all tissues and in bovine adrenocortical cells even after many population doublings in culture. In contrast to the specific and rapid demethylation of sites in CYP17, satellite I shows a slower and apparently random loss of methylation that extends over the entire replicative life span. These changes in methylation provide examples of genetic instability in cells that undergo senescence in culture. Future experiments will focus on the relationship of these events to the phenotypic switching process.