Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)