We studied the fourth component of human complement (C4) allotypes in 58 Japanese individuals. The technique of Southern, with C4 and 21-OH cDNA probes, was used to examine the genomic DNA of 45 individuals typed for C4 by protein electrophoresis. Novel HindIII C4 10- and 5-kb and EcoRI C4 13-Kb restriction fragments were identified in each of nine Japanese individuals. The novel fragments were different from the previously described C4B long (HindIII 31-kb, TaqI 6-kb, BamHI 4.3-kb, and EcoRI 12-kb) and C4B short (HindIII 25-kb, TaqI 5.4-kb, BamHI 3.5-kb, and EcoRI 15-kb) fragments. All novel HindIII- and EcoRI-positive individuals carried C4B5, BfS, and HLA-Bw54. Therefore, the fragments were characteristic for the C4B5 allele. The C4 region was analyzed to determine the restriction sites by single and double digests of uncloned genomic DNA with several restriction endonucleases. It is speculated that an insertion gene lies between the 3' end of the 21-OH and the 5' end of the C4B genes.