In chronically implanted awake rats, microinjections of chlordiazepoxide (5 x 10(-7) M) into the dorsal raphé significantly attenuated the inhibition of lever-pressing for food elicited by a signal of punishment. This effect is abolished by prior application of 5,7-dihydroxytryptamine into the dorsal raphé (3 weeks after the infusion of the neurotoxin, dorsal raphé tryptophan hydroxylase activity was reduced to 25% of control values). Furthermore, the disinhibitory effect of intra raphé chlordiazepoxide can be mimicked or potentiated by intra raphé dorsalis application of serotonin (10(-7) or 10(-8) M, respectively). Further evidence for a crucial interaction between benzodiazepines and serotoninergic processes are provided by in vitro experiments showing that chlordiazepoxide or diazepam (10(-5) M) are able to facilitate the K+-evoked [3H]serotonin release from rat midbrain slices. Finally, a high density of [3H]flunitrazepam binding sites was found in the dorsal (and the median) raphé nucleus, the Kd and Bmax values being not altered by prior infusion of 5,7-dihydroxytryptamine. These in vitro data suggest possible means by which intra raphé (and perhaps peripherally administered) benzodiazepines may affect the activity of serotoninergic neurons and thereby produce their effects on experimental anxiety.