Recent experiments have suggested the existence of excitatory amino acid (EAA)-containing afferents to the median raphe nucleus. In the present study we investigated the functional significance of EAAs in the median raphe (MR) by examining the behavioral and biochemical effects of intra-raphe injections of EAA antagonists. Injections of kynurenic acid, gamma-glutamylglycine, 2-amino-5-phosphonovaleric acid (2-APV) and 2-amino-7-phosphonoheptanoic acid (2-APH) into the median raphe resulted in marked hyperactivity. In contrast to the effect of 2-APV and 2-APH, intra-raphe injections of the homologues of these compounds with 4, 6 or 8 carbon atoms, which have a lower affinity for excitatory amino acid receptors, were without significant effects on activity. Additionally, the effects of 2-APV were shown to be stereospecific to the active D-isomer further suggesting receptor mediation of the effect. Injections of EAA antagonists into the dorsal raphe nucleus or the ventral tegmental area were much less effective in increasing activity than were injections into the MR, suggesting anatomical specificity of the effect. Injections of 2-APV into the median raphe were also shown to result in a reduction of serotonin metabolism within the hippocampus and an increase in dopamine metabolism within the nucleus accumbens and the magnitude of both of these effects was positively correlated with the behavioral responses to the injections. These findings suggest that cells within the median raphe may be subject to a tonic excitation exerted through EAA receptors.