The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching.