The radioiodinated pindolol analogs 125I-labeled cyanopindolol ([125I]CYP) and 125I-labeled hydroxybenzylpindolol ([125I]HBP) have been used to study binding to human platelet beta-adrenergic receptors. [125I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (Kd) of 14 +/- 3 pM and maximal binding capacity (Bmax) of 18 +/- 4 fmol/mg protein. Binding of [125I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8 . 10(7) s-1 . M-1 and 3.8 . 10(-4) s-1, respectively. [125I]HBP binds to a saturable class of platelet membrane sites with a Kd of 50 +/- 10 pM and Bmax of 32 +/- 6 fmol/mg protein. [125I]HBP also binds to a saturable class of sites on intact platelets with a Kd of 58 +/- 14 pM and Bmax of 24 +/- 4 molecules per platelet. Binding of [125I]CYP and [125I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (-) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol much greater than isoproterenol greater than epinephrine greater than practolol greater than norepinephrine greater than phenylephrine. These observations indicate that [125I]CYP and [125I]HBP bind to platelet sites which have the pharmacological characteristics of beta-adrenergic receptors but which are not typical of either the beta 1 or beta 2 sub-type.