In unanesthetized spinal cats, clonidine HCl (5-50 microgram/kg, i.v.) rapidly and markedly depressed excitatory transmission through two spinal pathways to sympathetic preganglionic neurons. Depression through either pathway was dose-dependent and persisted for more than 3 hr but could be rapidly antagonized at any stage by tolazoline HCl in a dose-ratio of about 1:100. The two spinal pathways were also depressed transiently by L-dopa and for prolonged periods by 5-HTP; both precursors were shown to act by releasing 5-HT from bulbospinal 5-HT terminals and their depressant effects were also antagonized by tolazoline. In the absence of 5-HT-induced depression, L-dopa only enhanced transmission through both pathways by inducing release of catecholamines from bulbospinal NE terminals. These results indicate that clonidine depresses sympathetic activity by stimulating inhibitory 5-HT receptors on sympathetic preganglionic neurons, a mechanism that adequately accounts for its central vasodepressor effect.