Intraspinal microstimulation ( ISMS) through a single microelectrode can induce locomotion in cats spinalized at T-13 1 wk before ( untrained) or after 3-5 wk of treadmill training. Here we study the optimal parameters of ISMS and the characteristics of locomotion evoked. ISMS was applied in the dorsal region of segments L-3-S-1 at different lateralities ( midline to 2.5 mm) and after an intravenous injection of clonidine ( noradrenergic agonist). Kinematics and electromyographic recordings were used to characterize locomotion. ISMS could induce a bilateral locomotor pattern similar to that obtained with perineal stimulation, and the characteristics of locomotion varied according to the spinal segment stimulated. Mechanisms by which ISMS could evoke locomotion were then investigated by stimulating, inactivating, or lesioning different spinal structures. Dorsal root stimulation ( DRS), just like ISMS, could evoke a variety of ipsi- and bilateral nonlocomotor movements as well as locomotor responses. This suggests that sensory afferent pathways are involved in the production of locomotion by ISMS. Microinjections of yohimbine ( noradrenergic antagonist) in L-3 and L-4 segments or a complete second spinal lesion at L-3-L-4 abolished all locomotor activity evoked by ISMS applied at more caudal segments. Progressive dorsoventral spinal lesions at L-3 or L-4 and restricted ventral lesions at L-4 further suggest that the integrity of the ventral or ventrolateral funiculi as well as the L-3-L-4 segments are critical for the induction of locomotion by ISMS at L-5 to S-1 or by DRS at these caudal segments.