Fetal hemoglobin production can be reactivated in vivo in adult persons with various hemoglobinopathies and other hemopoietic disorders, and in cultures of adult erythroid progenitors. We show that the activation of Hb F in adult cells is transcriptional in nature and is accompanied by the appearance of DNase I-hypersensitive sites and undermethylation of Hpa II sites 5' to the gamma-globin genes. Production of Hb F in culture is strongly modulated by the environment, and the repression of Hb F synthesis by specific culture conditions has been reported. By nuclear runoff, chromatin, and methylation analyses, we show that this inhibition of Hb F production in vitro is at the level of transcription with the concomitant loss of characteristic gamma hypersensitive sites and methylation of gamma Hpa II sites. These data indicate, first, that the organization of globin chromatin of adult cells that produce fetal hemoglobin resembles that of fetal erythroid cells and, second, that this organization switches from a fetal to an adult pattern in response to changes in the environment of the erythroid cells.