The specific binding of (3H)yohimbine to alpha 2 adrenoceptors in rat cerebral cortex varied significantly with methods of membrane preparation. Membranes prepared in a sucrose containing buffer showed markedly lower Bmax values than those prepared in hypotonic buffer without any change in affinity (Kd) for (3H)yohimbine. Higher concentrations of residual endogenous norepinephrine were found in sucrose prepared membranes. In the presence of exogenous norepinephrine (10(-8), 10(-7) M), membranes prepared in hypotonic buffer showed apparent reduced receptor densities similar to those observed in sucrose prepared cerebral membranes. The presence of exogenous norepinephrine did not produce any apparent change in the overall Kd for (3H)yohimbine. The depression of (3H)yohimbine binding capacities could, in all cases, be reversed by performing incubations in the presence of 200 mM NaCl and 10 microM Gpp(NH)p which synergistically dramatically reduce the affinity of norepinephrine for cerebral alpha 2 adrenoceptors. It is concluded, therefore, that the lower (3H)yohimbine binding capacities in sucrose prepared membranes appears to be due to the occupancy of receptor sites by residual norepinephrine in a pseudo non competitive manner. The reduced affinity of retained endogenous norepinephrine for the alpha 2 adrenoceptor, in the presence of Na+ and guanine nucleotides, would seem to be the cause of the apparent increase in (3H)yohimbine binding sites produced by these modulators in sucrose prepared membranes.