Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R., and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154--161). Dissociation of specific (-)-[3H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37 degrees C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (-)-[3H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((-)-isoproterenol) or beta-adrenergic antagonist ((-)-alprenolol or (-)-propanalol). Neither agonists nor antagonists influenced the rate of (-)-[3H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37 degrees C. Although analysis of the steady-state binding of (-)-[3H]dihydroalprenolol to fat cell membranes yields Hill coefficients, nH, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.