A multiple protein-DNA complex formed at a human alpha-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic zeta-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human zeta-globin promoter in fetal and adult transgenic mice. Furthermore, zeta-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human zeta-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.