Serotonin reveals ineffective (subthreshold) pathways from the C2 lateral funiculus to ipsilateral phrenic motoneurons in spinalized rats. The objective of the present study was to investigate serotonergic modulation of crossed-spinal pathways to contralateral phrenic motoneurons. Rats (n = 10) were anesthetized (urethane), paralyzed, vagotomized, and artificially ventilated. The spinal cord was hemisected at C1-C2 and, on the intact side, a tungsten stimulating electrode was placed ventral to the C2 dorsal root entry zone in the dorsolateral (approximately 1.1 mm) or the ventrolateral funiculus (approximately 2.2 mm depth). Single shocks (100-750 microA, 0.1-0.5 ms, 2 Hz) elicited a short-latency (approximately 1.0 ms to peak) excitation in the ipsilateral phrenic nerve, but usually evoked little or no response in the contralateral phrenic nerve at either stimulus site. Following systemic injection of the monoamine oxidase inhibitor pargyline (25 mg/kg) and the serotonin precursor 5-hydroxytryptophan (5-10 mg/kg), complex responses were revealed in the contralateral phrenic nerve, including: (1) spontaneous tonic activity; (2) a short-latency (approximately 1.0 ms to peak) evoked excitation; and (3) two long-latency (approximately 2.2 and 7.8 ms to peak) evoked excitations. The longest latency excitation was expressed only when the stimulating electrode was positioned in the dorsolateral funiculus. Contralateral evoked responses were blocked by systemic methysergide (2-6 mg/kg), a broad-spectrum serotonin receptor antagonist. These results indicate that serotonin converts ineffective crossed phrenic pathways in the spinal cord to effective pathways. It remains to be determined whether serotonin is both necessary and sufficient in this modulatory process, or if it is a nonspecific result of increased phrenic motoneuron excitability.