The evolutionary relationship between two central major histocompatibility complex (MHC) genes, C4 and CYP21, was investigated by employing pulsed field gel electrophoresis (PFGE) and conventional restriction fragment length polymorphism (RFLP) analyses in human and nonhuman primates. Using Taq I in conjunction with C4 and CYP21 probes, it has been found that there are four major types of C4 genes [defined by 7.0, 6.4, 6.0, and 5.4 kilobases (kb) Taq I fragments] and two major types of CYP21 genes (3.7 and 3.2 kb fragments) in human and nonhuman primates including chimpanzee, gorilla, and orangutan. All of the eight possible combinations of C4 and CYP21 genes can be identified on one or more human ancestral haplotypes (AH). It is concluded that each of the major types of C4 and CYP21 (and each of the combinations between these) predated human speciation. PFGE analysis with Mlu I and Pvu I suggested that each C4+CYP21 segment has a specific length of 30-50 kb and that each AH carries one, two, three, or even more segments. In the case of C4, it is important to note that there is no simple relationship between the RFLP and the protein classifications. Thus, at least some of the expressed polymorphisms could be relatively recent in that they are carried by the same or different gene types. These findings are consistent with the hypothesis that MHC AHs have been formed from a large pool of specific genomic segments and that further haplospecific polymorphism has developed subsequently.