zeta-Globin is the embryonic form of the alpha chain of hemoglobin. Transgenic mice generated with zeta-globin constructs containing the zeta-globin gene, 557 bp of 5′ flanking sequence, and 2-kb of 3′ flanking sequence linked to the beta-globin locus control region hypersensitive site 2 (HS2) expressed human zeta-globin only in embryonic yolk sac erythroid tissue, and not in definitive erythroid tissue in the fetal liver or in adult peripheral blood. To determine what sequences in the 5′ flanking region of the zeta-globin gene might be important for developmental specificity, a series of 5′ deletion constructs of the zeta-globin gene were made and used to generate transgenic mice. The 5′ ends of these constructs were located 417, 207, and 128 bp 5′ to the zeta-globin transcriptional start site, and HS2 was included to increase the level of erythroid-specific expression. In all lines of mice tested, human zeta-globin was expressed only in embryonic tissue, and not in fetal livers or in adult peripheral blood. Expression was independent of copy number and appeared to be dependent on the site of transgene insertion. These data suggest that the proximal 128 bp of the zeta-globin promoter is sufficient to properly regulate zeta-globin expression during development.