To begin to study the sequence variations identified in the 5' flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple transcription initiation sites. In vitro DNAseI footprinting analyses revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Transfection of ankyrin promoter/reporter plasmids into tissue culture cell lines yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody supershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In transfection assays, mutation of the Sp1 site had no effect on reporter gene expression, mutation of the CACCC site decreased expression by half, and mutation of the GATA-1 site completely abolished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BKLF.