Four rat × mouse hybridomas secreting monoclonal anti-idiotypic (anti-Id) antibodies (MAb) specific for the transgene-encoded antibody of the 207–4 transgenic mouse line, which carries the V_H1/V_κ24 gene segments of the IgA, phosphocholine-(PC) specific MOPC167 myeloma, were developed from a fusion of Ag8-X63.653 mouse cells with spleen cells from a rat immunized with MOPC167 and HPCM27 anti-PC antibodies. The anti-Id MAb were shown by ELISA to be specific for PC-binding proteins of V_H1/Vκ24 H and L chains of various isotypes. They did not bind V_H1/V_κ22, V_H1/V_κ8, or V_H1/V_κ1 PC-binding proteins or other IgA or IgM myeloma proteins. Analysis by flow cytometry demonstrated that these MAb bind to the transgene-encoded membrane immunoglobulin (sIgM) as expressed on >95% of the B220 positive 207–4 spleen cells. All four MAb were able to inhibit the binding of MOPC167 to PC conjugated to bovine serum albumin. Differences in fine specificity of binding were demonstrated by differential staining of spleen cells of the 216–7 μκδMem MOPC167 transgenic mice. In these mice endogenous H chains associate with the transgene encoded L chain to form MOPC167 crossreactive idiotopes. Two of the MAb, 28–4–3 and 28–6–20, stained significant numbers of cells, while MAb 28–5–15 did not bind to 216–7 cells. Three of the MAb, 28–5–15,28–6–20, and 28–4–3, when conjugated to Sepharose beads, were able to induce DNA synthesis in cultures of 207–4 transgenic spleen cells. None of the MAb were able to induce an antibody response in vivo. These MAb should prove useful in staining PC-transgenic B cells for flow cytometry studies and in defining early cellular events in the activation of idiotype positive B cells by anti-Id antibodies.