Abstract The activity of interneurons monosynaptically excited by 1a afferents and mediating the reciprocal inhibition of motoneurons of the antagonistic muscles was recorded in mesencephalic cats. The activity was studied during locomotion evoked by stimulation of the ‘locomotor region’ of the mesencephalon. The neurons turned out to be active in a definite phase of the step simultaneously with that muscle which supplied them by 1a afferent input. In preparations with de-efferent hindlimbs, stimulation of the ‘locomotor region’ evoked a periodic process in the spinal cord (‘fictive’ locomotion); in this case bursts of activity in the interneurons also coincided with the activity of motoneurons of corresponding muscles. Under such conditions excitation of 1a afferents by the passive stretch of the muscle or by electric stimulation of the muscle nerve resulted in an increase of the activity of interneurons, which depended on the phase of the cycle. The data obtained show that during locomotion there are at least two sources of inhibition of motoneurons of antagonistic muscles: (i) the activity of 1a afferents of the active muscle, mediating by corresponding interneurons; and (ii) signals coming through the same interneurons from the central mechanisms generating stepping movements.