C4b-binding protein (C4BP) is an important component in the regulation of the complement system and also binds the anticoagulant vitamin K-dependent protein S. These activities are performed by distinct, although structurally related, polypeptides of 70 kDa (alpha chain) and 45 kDa (beta chain), respectively. In this report we have investigated the genetic relationships between these polypeptides. Using pulsed field gel electrophoresis analysis we demonstrate that the genes coding for the alpha (C4BP alpha) and beta (C4BP beta) chains are closely linked within the regulator of complement activation gene cluster. In addition, we have determined that the 3' end of the C4BP beta gene lies 3.5-5 kilobases from the 5' end of the C4BP alpha gene. These findings support the concept that the C4BP alpha and C4BP beta genes are the result of a gene duplication event.