Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian natural killer (NK) cells. In the present study an anti-idiotypic monoclonal antibody (mAb 7D12) was generated against idiotopes on an mAb (mAb 6D3.2) that recognizes a putative receptor on NCC. The idiotypic specificity of mAb 7D12 was determined in competition assays by incubating biotinylated mAb 7D12 with mAb 6D3.2 hybridoma cells following preincubation with combinations of biotinylated 7D12 with either nonbiotinylated homologous or heterologous mAb. The ligand recognized by mAb 7D12 (determined by flow cytometry) was found on cells from the anterior kidney, spleen, thymus, PBL, liver, and brain. NCC lysis of IM-9 targets was inhibited 76% following preincubation of the target cells with different concentrations of mAb 7D12. The involvement of the ligand recognized by mAb 7D12 in the NCC lytic cycle was determined by showing that this mAb produced 50% inhibition of NCC conjugate formation with NC-37 target cells. Biochemical analysis using SDS-PAGE and Western blotting revealed that mAb 7D12 recognized 54 and 65 M(r) proteins in IM-9 target cell lysates. These studies demonstrated that an idiotope on a NCC specific anti-receptor mAb was an "internal image" of a target cell ligand. The anti-id mAb generated against this image (idiotope) inhibited NCC cytotoxicity and thus was equivalent to an NCC receptor that binds to a target cell ligand involved in NCC recognition.