Immunization of random bred Charles River CD strain (CR) rats with poly Glu52Lys33Tyr15 (no. 3) in Freund's complete adjuvant resulted in a highly variable antibody response which was most likely due to random genetic fluctuations in the population. Immunization of six inbred (BN, BUF, F344, WF, LEW and ACI) and one systematically random bred (WIST) strain of rats with a variety of synthetic polypeptide antigens resulted in a reproducible antibody response in all strains, but with marked interstrain differences. Studies with (ACI × F344) F1 hybrid rats showed that the ability to make antibody was transmitted as a complex genetic control mechanism which probably involved several genes. The presence of tyrosine in the antigen was not necessary in order to elicit an antibody response, and its effect on the magnitude of the antibody response was variable and strain-dependent. All strains responded to a D-amino acid polypeptide and showed interstrain differences. Aggregation of an L-polypeptide with methylated bovine serum albumin elicited an increased antibody response in those strains which produced a low titer to the polypeptide alone and reduced interstrain differences in the response. A D-polypeptide aggregate elicited more antibody in all strains except WIST and abolished the interstrain differences in the antibody response. The antibody response to a spatially ordered, dinitrophenylene (DPE) cross-linked polypeptide was higher, showed increased interstrain differences and showed a different specificity than the antibody response to the parent, linear polypeptide or the (Lys-DPE) and DNP conjugates, which are partial structural analogues of the DPE cross-link. Thus, the DPE cross-linked polypeptide had unique antigenic sites which depended upon the spatial organization of the molecule. In contrast, the presence of amide cross-links in the molecule introduced a relatively small change in immunogenicity and antigenic specificity. The studies reported here provide evidence that the effects of the various structural aspects of an antigen on immunogenicity and antigenic specificity are under genetic control. The ability to respond to a particular facet of antigenic structure probably involves several genes.