We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde I, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLPs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu II site in the fifth intron.