Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.