We previously identified the murine homologue of the human β-globin Locus Control Region (LCR) 5′ HS-2. The λ clone containing murine 5′ HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5′ HS-3 is located approximately 16.0 kb upstream from the mouse εy-globin gene, but no region homologous to human 5′ HS-4 was present in our clone. Using a reporter system consisting of a human γ-globin promoter driving the neomycin phosphotransferase gene (γ-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the εy-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5′ HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5′ HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5′ HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5′ HS-3: first, a DNA fragment containing 5′ HS-3 confers copy number-dependent, integration-site independent inducibility on a linked β-globin gene in the MEL cell environment. Secondly, a strong DNAsel hypersensitive site maps to the location of the 5′ HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5′ HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5′ HS-3 may therefore contain information that contributes to the development-specific expression of the β-like globin genes.