The anatomical substrate and behavioral pharmacology of stimulation-produced analgesia resulting from electrical stimulation of the pontomesencephalic nucleus cuneiformis (NCF) was determined in the present study. Maximum increase in nociceptive tail-flick latencies following NCF stimulation occurred during the first 5 min post stimulation and decreased afterwards. The increased reflex latency could be attenuated by prior treatment with the narcotic antagonist, naloxone or the cholinergic antagonist, scopolamine. The anatomical projections of NCF were identified in autoradiographic and histochemical studies. Ipsilateral fibers coursed caudal from the NCF injection site through the ventral pontine reticular formation to innervate nucleus raphe magnus and the ipsilateral nucleus magnocellularis. At rostral medullary levels fibers coursed dorsolateral to innervate the ipsilateral nucleus reticularis parvocellularis. Descending contralateral fibers crossed through the decussation of the superior cerebellar peduncle, then coursed ventrolaterally projecting to the contralateral nucleus magnocellularis. Two primary groups of ascending fibers were observed. The dorsally located group ascended through the central tegmental tract projecting to the dorsal raphe, ipsilateral periaqueductal gray, nucleus parafascicularis and centromedianus, the intermediolateral and lateral thalamic nuclei. The ventral group coursed ventrolateral from the injection site projecting to the substantia nigra, zona compacta, ventral tegmental area of Tsai, zona incerta, Fields of Forel, lateral hypothalamic nucleus and nucleus reuniens. These anatomic and behavioral data suggest that NCF plays an important role in sensory/motor integration relevant to pain transmission.