Erythroid cells regulate heme biosynthesis in a manner that is distinct from all other cell types. While heme negatively regulates the synthesis of the housekeeping delta-aminolevulinate synthase (ALAS-N) in all non-erythroid cells, the expression of an erythroid-specific isozyme (ALAS-E) is developmentally regulated in red blood cells. As a first step towards understanding the molecular basis for the transcriptional regulation of ALAS-E during erythropoiesis, we cloned and characterized the chicken ALAS-E locus. This gene spans 18 kbp and is composed of eleven exons. The intron/exon structure of erythroid ALAS was found to be conserved among several vertebrate species. Direct RNA sequencing identified a 5' untranslated region that is derived from two continuous exons and is predicted to form a very stable stem-loop structure that bears resemblance to the ferritin iron-responsive element. Tissue-specific expression of the ALAS-E gene was analyzed by transient transfection assays in hematopoietic cells of both erythroid and non-erythroid origins. These experiments identified distal (-784 to -505 bp) and proximal (-155 to +21 bp) promoter elements which are required for high level, erythroid-specific transcription.