In an effort to understand how the heme biosynthetic pathway is uniquely regulated in erythroid cells, we examined the structure of the gene encoding murine delta-aminolevulinate dehydratase (ALAD; EC18.104.22.168), which is the second enzyme of the pathway. The gene contains two first exons, named 1A and 1B, which are alternatively spliced to exon 2, where the coding region begins. Each first exon has its own promoter. The promoter driving exon 1A expression is TATA-less and contains many GC boxes. In contrast, the exon 1B promoter bears regulatory sequences similar to those found for beta-globin and other erythroid-specific genes. Tissue distribution studies reveal that ALAD mRNA containing axon 1A is ubiquitous, whereas mRNA containing axon 1B is found only in erythroid tissues. This finding, together with our further observation that GATA-1 mRNA levels increase 3-fold during maturation of murine erythroid progenitor cells, may help explain simultaneous 3-fold increases in exon 1B expression. The unexpected result that axon 1A expression also increases 3-fold during CFU-E maturation may be attributable to the action of NF-E2, since there is a potential binding site in a position analogous to the NF-E2 site in the locus control region of the beta-globin gene cluster.