The potential usefulness of internal-image anti-idiotype antibodies (Ab2s) in modulating hosts' immune responses to tumor-associated antigen (TAA) have stimulated considerable interest in the development and characterization of Ab2s. Six different mouse monoclonal Ab2s (NUH31, 44, 51, 71, 82 and 91) were generated against murine monoclonal antibody G250 (MAbG250) which recognizes a human renal-cell carcinoma-associated antigen. All 6 Ab2s showed specificity for the MAbG250 paratope in Western-blot analysis. In inhibition assays, all Ab2s were able to compete with the nominal antigen, albeit with differing efficiency. Based on cross-blocking studies for idiotope mapping, the Ab2s could be divided into 2 groups (group I; NUH31, 51, 71, group 2; NUH44, 82, 91). However, cross-reactivity between these 2 groups was observed, indicating that they recognized partly overlapping epitopes on the paratope of MAbG250. Sera from rabbits immunized with Ab2s showed reactivity with G250 antigen-positive cell lysates, but not with antigen-negative cell lysates. Additional studies revealed that all Ab2s were able to induce anti-anti-idiotype antibodies resembling MAbG250 (Ab1'). These findings suggest that the Ab2s functionally mimic the original G250 antigen and may be of use in the immunotherapy of human renal-cell carcinoma.