[3H]Diazepam appears to bind specifically to a single, saturable, binding site located on rat brain membranes, with an affinity constant near 3 nM at pH 7.4. Specific binding constitutes more than 90% of total binding at 0 degrees and less than 10% of total binding at 37 degrees. Arrhenius plots suggest a sharp conformational change in the diazepam receptor near 18 degrees. Mitochondrial fractions from rat kidney, liver, and lung exhibit some [3H]diazepam binding that can be displaced by nonradioactive diazepam and several other benzodiazepines. However, Ro-4864, which is almost inactive in displacing [3H]diazepam from brain membranes, is extremely potent in displacing it from kidney mitochondria. Conversely, clonazepam, the most potent inhibitor of brain binding, is an extremely weak inhibitor of kidney binding. Furthermore, diazepam binding to kidney mitochondria has an affinity constantof 40 nM, about 15 times higher than that in brain. No specific diazepam binding was detected in intestine or skeletal muscle. Thus, specific [3H]diazepam binding to membranes appears to be restricted to brain, where it is unevenly distributed: the density of diazepam receptors is about five times higher in cortex (the highest density) than in pons-meddula (lowest density). Trypsin and chymotrypsin completely abolished specific [3H]diazepambinding in brain and kidney.