A major positive regulatory element has recently been identified 40 kb upstream from the human ζ2-globin gene. This regulatory element increases the expression of a linked α-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the β-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (αLCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This αLCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the α-globin gene, some increase in activity is seen when structural elements from the 5' end of the α-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the αLCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease α-globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the αLCR. These studies suggest a role for the αLCR in the basal activity of the α-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of α-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the αLCR.