The neuronal pathways involved in the muscle relaxant effect of tizanidine were examined by measurement of spinal reflexes in rats. Tizanidine (i.v. and intra-4th ventricular injection) decreased the mono- and disynaptic (the fastest polysynaptic) reflexes (MSR and DSR, respectively) in non-spinalized rats. Depletion of central noradrenaline by 6-hydroxydopamine abolished the depressant effect of tizanidine on the MSR almost completely and attenuated the effect on the DSR. Co-depletion of serotonin by 5,6-dihydroxytryptamine and noradrenaline resulted in more prominent attenuation of tizanidine-induced inhibition of the DSR. Supraspinal receptors were then studied using yohimbine- and some imidazoline-receptor ligands containing an imidazoline moiety. Idazoxan (I₁, I₂, I₃, and α₂), efaroxan (I₁, I₃, and α₂), and RX821002 (I₃ and α₂), but not yohimbine, an α₂-adrenergic receptor antagonist with no affinity for I receptors, antagonized the inhibitory effects of tizanidine. Thus, supraspinal I receptors (most likely I₃) and descending monoaminergic influences are necessary for tizanidine-induced inhibition of spinal segmental reflexes.