The pretreatment of rat cerebral cortical membranes with 10 or 100 microM isoproterenol at 37 degrees C for 40 min caused a significant elevation of the Bmax value of [3H]clonidine binding but pretreatment at 4 degrees C did not affect the value. The isoproterenol-induced increase in the Bmax value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the Bmax value of [3H]dihydroalprenolol binding but neither Kd nor Bmax of [3H]WB 4101 binding was affected by this treatment. Fifty microM propranolol or 100 microM GTP produced a significant reduction in isoproterenol-induced elevation of the Bmax value of [3H]clonidine binding. In contrast, 100 microM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only Bmax value in high affinity binding of [3H]clonidine was increased by isoproterenol. It is suggested that isoproterenol increases the density of alpha 2-adrenoceptors in a temperature-dependent manner. The direct interaction between beta- and alpha 2-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.