We have identified a previously undetected cis-acting element in the mouse β-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the β-globin direct-repeat element (βDRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The βDRE motif is highly conserved in all adult mammalian β-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the βDRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the βDRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in βDRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the βDRE have implications for the mechanism of action of the element in regulating β-globin transcription and for mutational studies of other repetitive or redundant transcription elements.