We have previously described a class of immunostimulatory ribonucleoside, exemplified by loxoribine (7-allyl-8-oxoguanosine) and 8-bromoguanosine, that act on cycling B cells to promote nonspecific proliferation and differention, and that synergize with Ag to recruit quiescent Ag-reactive B cells to undergo differentiation and high level Ab production. In murine B cells, two distinct binding activities have been characterized that have dissociation constants that parallel their distinctive dose-response profiles. The SJL mouse strain, which is hypo-responsive to the B cell proliferative properties of these molecules, exhibits a dissociation constant of 10- to 20-fold lower affinity than that of normal murine strains; its Kd for the higher affinity-binding interaction, however, is essentially normal. These observations suggested the existence of two distinct binding activities. Recently, immunoselective subgroups of loxoribine have been described, with an ability to stimulate individual components of the immune response that correlates with specific changes in discrete domains of the nucleoside molecule. By using a panel of nonstimulatory loxoribine analogues as inhibitors, we now report further evidence for the existence of unique sites on loxoribine binding proteins that recognize distinct domains on nucleoside analogues; changes in these regions confer immunologic selectivity. These data confirm the conclusions of previous studies, extend our understanding of the topology of subsites that are otherwise silent, and provide insight into the binding site mediating adjuvanticity. These results allow more profound appreciation of the nature of structural changes that will permit a tighter fit between ligand and binding site, ultimately promoting the design of more potent immunoselective agents.