Selected chicken populations were analyzed by restriction fragment length polymorphism (RFLP) with a chicken MHC Class I (B-F) cDNA probe. The 13 highly inbred chicken lines differed in genetic origin and in MHC (B) haplotype, as distinguished by using hemagglutination with antisera against B-G and B-F antigens. The S1 sublines differed for B haplotype and antibody response to a synthetic polypeptide, GAT. In the highly inbred lines, band-sharing between lines from different origins was less than that between lines from same origin, showing the influence of the genetic background on chicken MHC Class I gene RFLP. In the S1 line, use of three restriction endonucleases (BglII, PvuII, and TaqI) produced MHC Class I RFLP that were associated with B haplotype, but not with immune response to GAT (IrGAT). A previous study in the authors' laboratory also demonstrated an association of MHC Class IIβ RFLP patterns with B haplotype, but not IrGAT, in the same line, suggesting that IrGAT is not controlled by MHC Class I or Class IIβ genes.