H‐2 class I‐negative, HLA‐A2.1‐transgenic HHD mice were used for a comparative evaluation of the immunogenicity of HLA‐A2.1‐restricted human tumor‐associated cytotoxic T lymphocyte (CTL) epitopes. A hierarchy was established among these peptides injected into mice in incomplete Freund's adjuvant which correlates globally with their capacity to bind and stabilize HLA‐A2.1 molecules. Co‐injection of a helper peptide enhanced most CTL responses. In contrast, classical HLA class I‐transgenic mice which still express their own class I molecules did not, in most cases, develop HLA‐A2.1‐restricted CTL responses under the same experimental conditions. Different monoepitope immunization strategies of acceptable clinical usage were compared in HHD mice. Recombinant Ty‐virus‐like particles, or DNA encoding epitopes fused to the hepatitis B virus middle envelope protein gave the best results. Using this latter approach and a melanoma‐based polyepitope construct, CTL responses against five distinct epitopes could be elicited simultaneously in a single animal. Thus, HHD mice provide a versatile animal model for preclinical evaluation of peptide‐based cancer immunotherapy.