An in vitro brainstem-spinal cord preparation from adult turtles was used to test the hypothesis that descending synaptic inputs to multifunctional spinal motoneurons (i.e., involved in respiration and locomotion) express activity-dependent depression or potentiation. The tissue was placed in a chamber that allowed for separate superfusion of the brainstem, spinal segments C(2)-C(4), and C(5)-D(1). Action potential conduction between the brainstem and spinal segments C(5)-D(1) was blocked by superfusing C(2)-C(4) with Na(+)-free solution. With C(5)-D(1) at [K(+)] = 10 mM, electrical stimulation at C(5) every 2 min evoked potentials in intact pectoralis (expiratory, inward rotation of shoulder) and serratus (inspiratory, outward rotation of shoulder) nerves that were stable for at least 2 hr. Application of conditioning stimulation (900 pulses at 1 or 10 Hz) at C(5) decreased pectoralis evoked potential amplitudes by approximately 40% initially and by 20% after 90 min; serratus evoked potentials were unaltered. Conditioning stimulation (100 Hz, 900 pulses) transiently depressed pectoralis evoked potential amplitude by <20% but produced a delayed 72% increase in serratus evoked potential amplitude after approximately 80 min. Conditioning stimulation (10 Hz) at C(5) also reduced the amplitude of sensory afferent evoked potentials in pectoralis produced by stimulating ipsilateral dorsal roots at C(8). Thus, long-lasting changes in descending synaptic inputs to multifunctional spinal motoneurons were frequency-dependent and heterosynaptic. We hypothesize that activity-dependent plasticity may modulate descending synaptic drive to spinal motoneurons involved in both respiration and locomotion.