Intraspinal microstimulation (ISMS) through a single microelectrode can induce locomotion in cats spinalized at T13 one week before (untrained) or after 3-5 weeks 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 L3-S1, at different lateralities (midline to 2.5 mm) and after an i.v. 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 ipsilateral and bilateral non-locomotor 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 L3 and L4 segments or a complete second spinal lesion at L3-L4 abolished all locomotor activity evoked by ISMS applied at more caudal segments. Progressive dorsoventral spinal lesions at L3 or L4 and restricted ventral lesions at L4 further suggest that the integrity of the ventral or ventrolateral funiculi as well as the L3-L4 segments are critical for the induction of locomotion by ISMS at L5 to S1 or by DRS at these caudal segments.