The fourth component of complement (C4) in man, is coded for by two separate but closely linked loci (C4A and C4B) within the major histocompatibility region (MHC), on the short arm of chromosome 6. Like class I and II loci of this region, the C4 genes are highly polymorphic with more than 30 alleles, including null alleles, assigned to the two loci. This extensive polymorphism, based mainly on electrophoretic mobility, provides a useful marker for studies of disease susceptibility. Several disorders, including systemic lupus erythematosus and type I diabetes, show associations with C4 phenotypes. We have used the technique of Southern with a C4 specific probe to examine the genomic DNA of individuals typed for C4 by protein electrophoresis. We have identified 10.7 and 3.8 kilobase (kb) BglII restriction fragments in each of 9 unrelated individuals with a C4A6 allele, and in none of 22 unrelated individuals in whom this allele was not expressed. This clear correlation of restriction fragment length polymorphism with C4 phenotype provides a precise basis for analysis of C4 polymorphism. It is likely to be of value in clinical investigations of autoimmune disease.