Locomotor stepping in the Nembutal-anesthetized rat was elicited by electrical stimulation of either of two sites in the right or left posterolateral hypothalamus. Essential midbrain loci were identified by reversibly blocking the elicited locomotion through local injections of the anesthetic procaine (15%, 0.5 microliter). Two types of critical midbrain sites were found. At ipsilateral block sites (n = 21), procaine blocked only that locomotion elicited by ipsilateral stimulation. These sites could be along the course of a direct descending ipsilateral pathway although a possible bidirectional pathway is not to be excluded. At bilateral block sites (n = 21), procaine blocked locomotion elicited by both ipsilateral and contralateral stimulation. These sites could be involved in functions prerequisite for the initiation of locomotion or in the generation of the stepping pattern. Procaine injections in 35 sites had no effect on locomotion. Ipsilateral and bilateral block sites were intermixed and generally located in regions ventral to the midbrain central gray: chiefly the anterior ventromedial midbrain, the pontis oralis nucleus and the pedunculopontine nucleus. Negative sites were located in both the dorsal and ventral midbrain. Ipsilateral block sites were relatively prevalent in the anterior midbrain, indicating that the locomotor initiation signals are lateralized at this level. Bilateral block sites were more prevalent in the posterior levels, suggesting that the initiation signals are proximal to, or interact with, circuits that have a bilateral influence on locomotion.