AMP deaminase (AMPD) is manifest through a multigene family in higher eukaryotes, including man. The human AMPD1 and AMPD3 genes have been cloned and partially characterized. This study describes the cloning, chromosomal localization, partial sequence and characterization of the human AMPD2 gene. Composed of nineteen exons and eighteen intervening sequences spanning nearly 14 kb of genomic DNA, the human AMPD2 gene is positioned on the short arm of chromosome 1 near the p13.3 boundary. Two alternative 5' exons (1A and 1B) are remotely located upstream, whereas the other seventeen are compressed into the 3' terminal one-half of the gene. Transient transfections of human retinal pigment epithelial (RPE) cells using heterologous constructs containing 5' flanking and 5' untranslated sequences cloned upstream of a luciferase reporter gene show that promoter activities are associated with exons 1A and 1B. Inspection of genomic DNA sequence reveals that AMPD2 promoter regions lack readily identifiable TATA boxes and are G + C-rich, particularly in the region of multiple transcription initiation sites in exon 1A. The regulation and evolution of the entire human AMPD multigene family are discussed.