Natural deletions of the region upstream of the human ␣-globin gene cluster, together with expression studies in cell lines and transgenic mice, identified a single element (HS ؊40) as necessary and perhaps sufficient for high-level expression of the ␣-globin genes. A similar element occupies the corresponding position upstream of the mouse (m) ␣-globin genes (mHS ؊26) and was thought to have similar functional properties. We knocked out mHS ؊26 by homologous recombination and observed the surprising result that instead of the expected severe ␣-thalassemia phenotype, the mice had a mild disease. Transcription levels of the mouse genes were reduced by about 50%, but homozygotes were healthy, with normal hemoglobin levels and only mild decreases in mean corpuscular volume and mean corpuscular hemoglobin. These results may indicate differences in the regulation of the ␣-globin clusters in mice and humans or that additional cis-acting elements remain to be characterized in one or both clusters. (Blood. 2002;100:3450-3456)