This study characterizes specific benzodiazepine receptors in rat brain membranes, showing high-affinity binding of [³H]diazepam with an affinity constant of approximately 3 nM at pH 7.4. Specific binding constitutes over 90% of total binding at 0°C and less than 10% at 37°C. Arrhenius plots suggest a conformational change in the receptor near 18°C. Mitochondrial fractions from rat kidney, liver, and lung show some [³H]diazepam binding that can be displaced by nonradioactive diazepam and other benzodiazepines. However, Ro-4864, which is 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 a weak inhibitor of kidney binding. The affinity constant for [³H]diazepam binding to kidney mitochondria is 40 nM, about 15 times higher than in brain. No specific [³H]diazepam binding was detected in intestine or skeletal muscle. Specific [³H]diazepam binding to membranes is restricted to brain, where it is unevenly distributed: the density of diazepam receptors is about five times higher in cortex than in pons-medulla. Trypsin and chymotrypsin completely abolished specific [³H]diazepam binding in brain and kidney. The study also describes distinct diazepam receptors in mitochondria from rat kidney, skeletal muscle, and lung. Specific [³H]diazepam binding to brain membranes is saturable with a half-maximal binding at about 3 nM. Nonspecific binding is about 7-9% of total binding. The binding site is probably a stable protein localized in cell membranes. Specific [³H]diazepam binding occurs to receptors in kidney, liver, and lung, but this binding is fundamentally different from that in brain membranes. The displacement characteristics of binding to kidney, liver, and lung are entirely different, and binding in kidney is associated with a mitochondrial fraction while that in brain is associated with a membrane fraction. The affinity of [³H]diazepam for its receptor in kidney is about 15 times less than in brain. The binding to serum albumin is clearly different pharmacologically from the binding to brain membranes. The results suggest that the [³H]diazepam binding site in brain is physiologically relevant, and an unknown endogenous transmitter acting on these receptors may exist.This study characterizes specific benzodiazepine receptors in rat brain membranes, showing high-affinity binding of [³H]diazepam with an affinity constant of approximately 3 nM at pH 7.4. Specific binding constitutes over 90% of total binding at 0°C and less than 10% at 37°C. Arrhenius plots suggest a conformational change in the receptor near 18°C. Mitochondrial fractions from rat kidney, liver, and lung show some [³H]diazepam binding that can be displaced by nonradioactive diazepam and other benzodiazepines. However, Ro-4864, which is 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 a weak inhibitor of kidney binding. The affinity constant for [³H]diazepam binding to kidney mitochondria is 40 nM, about 15 times higher than in brain. No specific [³H]diazepam binding was detected in intestine or skeletal muscle. Specific [³H]diazepam binding to membranes is restricted to brain, where it is unevenly distributed: the density of diazepam receptors is about five times higher in cortex than in pons-medulla. Trypsin and chymotrypsin completely abolished specific [³H]diazepam binding in brain and kidney. The study also describes distinct diazepam receptors in mitochondria from rat kidney, skeletal muscle, and lung. Specific [³H]diazepam binding to brain membranes is saturable with a half-maximal binding at about 3 nM. Nonspecific binding is about 7-9% of total binding. The binding site is probably a stable protein localized in cell membranes. Specific [³H]diazepam binding occurs to receptors in kidney, liver, and lung, but this binding is fundamentally different from that in brain membranes. The displacement characteristics of binding to kidney, liver, and lung are entirely different, and binding in kidney is associated with a mitochondrial fraction while that in brain is associated with a membrane fraction. The affinity of [³H]diazepam for its receptor in kidney is about 15 times less than in brain. The binding to serum albumin is clearly different pharmacologically from the binding to brain membranes. The results suggest that the [³H]diazepam binding site in brain is physiologically relevant, and an unknown endogenous transmitter acting on these receptors may exist.