The importance of the lateral habenula-dorsal raphe pathway in the control of in vivo [3H]serotonin release in the cat basal ganglia was examined using the push-pull cannula technique and an isotopic method for the estimation of [3H]serotonin continuously formed from [3H]tryptophan. [3H]Serotonin was measured in both caudate nuclei and substantiae nigra and, in some cases, in the dorsal raphe. Electrical stimulation of the lateral habenula decreased [3H]serotonin release in all structures studied. Blockade of the GABA inhibitory pathway to the lateral habenula by the local application of picrotoxin reduced [3H]serotonin release in both substantiae nigra and increased release of the 3H-amine in the dorsal raphe but was without effect on [3H]serotonin release in either caudate nucleus. This inhibition of nigral [3H]serotonin release was antagonized by simultaneous application of picrotoxin to the dorsal raphe. Substance P delivery to the dorsal raphe produced the same effects on [3H]serotonin release as described for picrotoxin application to the lateral habenula except that inhibition of nigral [3H]serotonin release was not prevented by local co-administration of picrotoxin. These results suggest that the lateral habenula can control serotonergic transmission in the basal ganglia and that this regulation may be different for those serotonergic neurons innervating the caudate nucleus versus those projecting to the substantia nigra.