The organization and physical linkage of four members of a major complement locus, the RCA locus, have been determined using the technique of pulsed field gradient gel electrophoresis in conjunction with Southern blotting. The genes encoding CR1, CR2, DAF, and C4bp were aligned in that order within a region of 750 kb. In addition, the 5' to 3' orientation of the CR1 gene (5' proximal to CR2) was determined using 5'- and 3'-specific DNA probes. The proximity of these genes may be related to structural and functional homologies of the protein products. Overall, a restriction map including 1,500 kb of DNA was prepared, and this map will be important for positioning of additional coding sequences within this region on the long arm of chromosome 1.