Isatin is an endogenous indole that influences a range of processes both in vivo and in vitro. It has a distinct and discontinuous distribution in the brain, as well as in other mammalian tissues and body fluids. However, the distribution of isatin binding sites in the brain is not known. Using a real-time β-imager we have investigated the distribution of [3H]isatin-specific binding in rat brain sections. The highest labeling was found in hypothalamic nuclei and in the cortex, hippocampus, and cerebellum. Administration of the mechanism based monoamine oxidase inhibitor, pargyline, reduced but did not abolish the specific binding of [3H]isatin in the rat brain. The distribution became cortex, cerebellum, hypothalamus > hippocampus > brain stem > thalamus ≈ striatum.