The binding of [3H]-Cyclopentyladenosine (CPA), an N6-substituted analog of adenosine, was examined in vitro. CPA bound with high affinity (Kd = 0.48 nmol/l) to rat brain membranes. Specific binding, which represented 90-97% of the total counts bound at a ligand concentration of 1 nmol/l, was saturable, reversible and sensitive to protein denaturation. The pharmacology of binding was consistent with the labeling of an A-1 receptor, the R- and S-diasteromers of N6-phenylisopropyladenosine (PIA) showing a sixteenfold difference in their ability to displace CPA. The prototypic A-1 selective adenosine agonist, N6-cyclohexyladenosine (CHA) was twofold less active than CPA in displacing radiolabeled CPA. Comparison of the ability of cold CHA and CPA to displace [3H]-CPA gave rate dissociation constants of 1.88 and 1.80 X 10(4) s-1, respectively suggesting that both CHA and CPA bound to the same recognition site. In contrast however, comparison of the binding of [3H]-CPA with that of [3H]-CHA showed distinct differences. The Kd for CHA was approximately twice that of CPA while the apparent Bmax was 60% greater. In comparing the pharmacology of CPA binding with that of CHA, it was found that CHA, S-PIA and the antagonist, PACPX were more active in displacing CHA than CPA. In general however, CPA has a binding profile very similar to that observed with CHA.