Polymorphisms within the Rh blood group system have been defined by serologic agglutination methods, but have not yet been defined at the DNA level. Two closely related genes associated with the Rh D antigen and with the Rh C/c and E/e antigens have been cloned. We used a Southern analysis incorporating probes to the 5′ and 3′ regions of the Rh C, E gene and D gene to identify polymorphisms associated with Rh C/c and E/e antigens, respectively. The D gene dosage could be determined by comparing the relative intensities of the D bands with bands from the 5′ and 3′ region of the Rh C, E gene. The concordance between restriction fragment length polymorphism (RFLP) patterns and serologic phenotypes for 102 randomly selected blood donors was 100% for C, e, and D, 94.8% for c, and 94.3% for E. The data are consistent with the sequences encoding the C/c epitopes residing on the 5′ side of those for the E/e epitopes. All samples discordant for the 3′ probe and E had the cE (r″) serotype. These data show that the gene coding for the cE serotype is different in Rh-positive and -negative individuals. The study demonstrates that Rh DNA typing, including D gene dosage measurements and Rh gene haplotyping, may supplement traditional serotyping methods in transfusion medicine.