We have recently reported the identification of a novel class of micromolar-affinity adenosine binding sites in rat brain membranes using the adenosine agonist 2-chloro[3H]adenosine (C1[3H]Ado). These binding sites are distinguishable from the A1 and A2 adenosine receptors by a number of pharmacological criteria, and we have designated this new class of binding sites as the A3 adenosine binding sites. In the present study, the effects of a wide range of divalent and trivalent cations on micromolar C1[3H]Ado binding to brain membranes were examined. Co2+, Ni2+ and La3+ markedly stimulated specific C1[3H]Ado binding by 45-150% above control when tested at concentrations of 1-10 mM. Ca2+ had no significant effect on binding except at high concentrations where it depressed binding slightly. Ca2+, however, completely prevented the stimulation of C1[3H]Ado binding by Co2+. These findings further distinguish the A3 class of adenosine binding sites from the previously characterized adenosine receptors and suggest that the A3 binding sites are associated with calcium systems in brain.