The spinal localization of the forelimb locomotor generators and their interactions with other spinal segments were investigated on in vitro brainstem-spinal cord preparations of new-born rats. Superfusion of the cervicothoracic cord (C1-T4) with high K+/low Mg2+ artificial cerebrospinal fluid (aCSF) evoked rhythmic motor root activity that was limited to low cervical (C7, C8) and high thoracic (T1) spinal levels. This activity consisted of synchronous, homolateral bursts and a typical alternating bilateral pattern. Rhythmic activity with similar locomotor-like characteristics could be induced with either serotonin (5-HT, 5 microm), N-methyl-d-aspartate (NMDA, 5 microm), kainate (10 microm) or a "cocktail" of 5-HT (5 microm) and NMDA (5 microm). During 5-HT/NMDA perfusion of the cervicothoracic cord, induced bursting was no longer restricted to C7-T1 levels, but also occurred at cervical C3-C5 levels and with C5-C8 homolateral alternation. Spinal transections between C6 and C7 cervical segments did not abolish rhythmic activity in C7-T1, but suppressed locomotor-like rhythmicity at C3-C5 levels. Reduced regions comprising the C7-C8 or C8-T1 segments maintained rhythmicity. Superfusion of the whole cord with 5-HT/NMDA induced ventral root bursting with similar frequencies at all recorded segments (cervical, thoracic and lumbar). After isolation, the T3-T10 cord was unable to sustain any rhythmic activity while cervical and lumbar segmental levels continued to burst, albeit at different frequencies. We also found that the faster caudal and the slower rostral locomotor generators interact to produce coordinated locomotor-like activity in all segments of the intact spinal cord. In conclusion, C7-T1 spinal levels display a strong motor rhythmogenic ability; with the lumbar generators, they contribute to coordinated rhythmic activity along the entire spinal cord of a quadrupedal locomoting mammal.