The mouse anti-idiotypic mAb (mAb2) 16D7 recognizes the paratope (combining site-associated epitope) of the syngeneic anti-human CD4 mAb HP2/6 (mAb1), a down-modulator of T cell function. 16D7 mimics CD4 in xenogeneic settings in humans and can thus be used to target CD4+ T cells for therapeutic purposes. To define the minimum structural requirement for CD4 mimicry, 16D7 isolated L and H chains were tested for their ability to inhibit mAb1 binding to either CD4 Ag or to mAb2. 16D7 L only specifically inhibited these interactions. Alignment of 16D7 L variable region sequence with that of CD4 defined peptides L2, L3 and L7 with at least 68% of similarity and 30% of identity to exposed regions of CD4. Furthermore, peptides L1, L4, L5, L6 and L8 were selected from a set of 10-mer overlapping peptides (covering the FW and CDR regions of 16D7 L) because of their reactivity with mAb1. Only the synthesized L2 reacted with HP2/6 in slot dot assay and inhibited mAb2-mAb1 interaction. Immunization of BALB/c mice with 16D7, 16D7-F(ab')2, 16D7 L and L2, resulted in the production of CD4-specific Ab3 only in mice immunized with 16D7 and its F(ab')2 fragments. The lack of CD4 reactivity with sera elicited with 16D7 L and L2 was not due to their lack of immunogenicity since both sera reacted with the corresponding immunogen and the former also inhibited the binding of mAb1 to 16D7. The results suggest that CD4 mimicry by 16D7 is based on a conformational epitope and L2 is only one of the HP2/6-specific contact points of 16D7.