1. Sulphorhodamine 101, a fluorescent dye and newly identified activity marker, was used to localize potential spinal locomotor networks in the neonatal rat spinal cord. 2. Preparations of the spinal cord with one entire hindlimb attached or the spinal cord in isolation were kept in vitro. Spinal locomotor activity was maintained chemically with NMDA (5-7.5 microM), in combination with 5-HT (7.5-20 microM), for 4-4.5 h in the presence of 0.0001-0.0005% sulphorhodamine 101. Matched non-locomoting controls were exposed to the dye in the absence of transmitters for a comparable time. Transverse sections of the lumbar spinal cord (L1-L6) were screened for rhodamine emission using an epifluorescence microscope. 3. In hindlimb-attached locomoting preparations with intact dorsal roots, labelled cells were found on the leg side in the dorsal horn (mainly laminae II-IV), in the intermediate grey (lamina VI-VII) and around the central canal (lamina X). Dorsal rhizotomy was performed on the leg side, to prevent synaptic activity due to afferent inflow. This largely reduced the number of labelled cells in the dorsal horn and in the lateral part of the intermediate grey matter. A further reduction of labelling in these areas was seen after complete isolation of the cord or when compared to the legless side, with the majority of labelled cells persisting in a bilateral cluster close to the central canal and in the medial intermediate grey. Few labelled cells were observed in non-locomoting preparations. The intensity of motoneuronal labelling was variable.(ABSTRACT TRUNCATED AT 250 WORDS)