In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.