HS‐26, the mouse homologue of HS‐40, is the major regulatory element of the mouse α‐globin gene locus. Like HS‐40, HS‐26 is located within an intron of a house‐keeping gene; comparison of the nucleotide sequences of HS‐26 and HS‐40 reveals conservation of the sequences and positions of several DNA binding motifs in the 5′ regions of both elements (3 GATA, 2 NFE‐2, and 1 CACCC sites) and the absence in HS‐26 of three CACCC sites and one GATA site that are present in the 3′ region of HS‐40, suggesting that the two elements might not be identical. We report here that when HS‐26 is linked to a 1.5 kb Psti human α‐globin gene fragment, it has a weak enhancer activity in induced MEL cells and in transgenic embryos, and it does not have any detectable activity in adult transgenic mice. This suggests that HS‐26 does not have Locus Control Region (LCR) activity but can act as an enhancer during the embryonic life when integrated at a permissive locus. To further test the importance of HS‐26 at its natural locus, we have generated embryonic stem cells and chimeric animals in which 350 bp containing HS‐26 have been replaced by a neomycin resistance gene by homologous recombination. The sizes of the chimeras' red cells were then estimated by measuring forward scattering on a FacsScan apparatus in hypotonic conditions. This revealed that a fraction of the chimeric animals' red cells were smaller than normal mouse red cells and were very similar to cells from mice heterozygous for α‐thalassemia. Density gradient analysis also suggested the presence of thalassemic cells. These results indicated that despite its lack of LCR activity, HS‐26 is important for the regulation of the mouse α‐globin gene locus. Am. J. Hematol. 54:30–39, 1997 © 1997 Wiley‐Liss, Inc.