Although locomotion is known to be generated by networks of spinal neurons, knowledge of the properties of these neurons is limited. Using neonatal transgenic mice that express enhanced green fluorescent protein (EGFP) driven by the c-fos promoter, we visualized EGFP-positive neurons in spinal cord slices from animals that were subjected to a locomotor task or drug cocktail [N-methyl-D-aspartate, serotonin (5-HT), dopamine, and acetylcholine (ACh)]. The activity-dependent expression of EGFP was also induced in dorsal root ganglion neurons with electrical stimulation of the neurons. Following 60-90 min of swimming, whole cell patch-clamp recordings were made from EGFP+ neurons in laminae VII, VIII, and X from slices of segments T(12) to L(4). The EGFP+ neurons (n = 55) could be classified into three types based on their responses to depolarizing step currents: single spike, phasic firing, and tonic firing. Membrane properties observed in these neurons include hyperpolarization-activated inward currents (29/55), postinhibitory rebound (11/55), and persistent-inward currents (31/55). Bath application of 10-40 microM 5-HT and/or ACh increased neuronal excitability or output with hyperpolarization of voltage threshold and changes in membrane potential. 5-HT also increased input resistance, reduced the afterhyperpolarization (AHP), and induced membrane oscillations, whereas ACh reduced the input resistance and increased the AHP. In this study, we demonstrate a new way of identifying neurons active in locomotion. Our results suggest that the EGFP+ neurons are a heterogeneous population of interneurons. The actions of 5-HT and ACh on these neurons provide insights into the neuronal properties modulated by these transmitters for generation of locomotion.