The mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23 bears the internal image of the determinant defined by the syngeneic immunizing anti-human high molecular weight melanoma-associated antigen (HMW-MAA) mAb 763.74, since it induces anti-HMW-MAA antibodies in syngeneic and xenogeneic hosts. To dissect the humoral immune response induced by mAb MK2-23 at the clonal level, 1351 hybridomas were generated from a BALB/c mouse immunized with mAb MK2-23. Serological and immunochemical assays showed that the anti-anti-idiotypic (anti-anti-id) mAb GH827, GH1002 and GH1081 react only with the immunizing anti-id mAb MK2-23 and that the anti-anti-id mAb GH149, GH368, GH464, GH518, GH586, GH704, GH786 and GH1151 react with both mAb MK2-23 and HMW-MAA. The eight HMW-MAA binding anti-anti-id mAb resembled mAb 763.74 in their reactivity patterns with a panel of cell lines, although the extent of reactivity was lower. Staining of surgically removed melanoma lesions detected subtle differences in the reactivity patterns of the eight HMW-MAA binding anti-anti-id mAb. This finding in conjunction with the differential ability of the eight anti-anti-id mAb to inhibit the binding of anti-HMW-MAA mAb 763.74 to melanoma cells and to mAb MK2-23 and with the different avidity of the binding to mAb MK2-23 suggest diversity in the fine specificity of the eight HMW-MAA binding anti-anti-id mAb. Like mAb 763.74, the eight HMW-MAA binding anti-anti-id mAb express the idiotopes recognized by the anti-id mAb MK2-23 and MK2-120. In contrast, the three anti-anti-id mAb GH827, GH1002 and GH1081, which do not bind HMW-MAA binding anti-anti-id mAb to the antibodies elicited by the membrane-bound HMW-MAA corroborates the validity of the use of anti-id mAb MK2-23 as an immunogen to implement active specific immunotherapy in patients with malignant melanoma.