The human genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol PI), corticosteroid-binding globulin (CBG), alpha 1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an approximately 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen-CBG-ATR-alpha 1 AT-KAL-PCI-AACT-tel. The genes occupy approximately 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by approximately 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies approximately 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is approximately 12 kb of intergenic DNA between KAL and PCI and approximately 19 kb between PCI and AACT. The proximal subcluster includes alpha 1AT, ATR, and CBG; it occupies approximately 90 kb of genomic DNA, with approximately 12 kb of DNA between alpha 1AT and ATR and approximately 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.