Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.