Commissural interneurons (CINs) send their axons across the midline to innervate contralateral targets and have been implicated in the coordination of left-right limb movements during locomotion. In the neonatal mouse spinal cord, we studied the firing properties and responses to serotonin (5-HT) of two classes of CINs: those whose axons turn caudally after crossing the midline (dCINs) and those whose axons bifurcate after crossing the midline (adCINs). During NMDA and 5-HT-induced locomotor-like activity, a majority of lumbar (L2) dCINs fired rhythmically with ventral root-recorded motor activity, although their firing phase was widely distributed throughout the locomotor cycle. In contrast, none of the adCINs fired rhythmically during fictive locomotion. We studied the baseline firing and membrane properties, and responses to current injection, in dCINs and adCINs that had been partially isolated by blockade of rapid synaptic transmission (with antagonists to glutamate, GABA, and glycine). No significant baseline differences were found between the cell types. In contrast, 5-HT significantly increased the excitability of the isolated dCINs by depolarizing the membrane potential, reducing the postspike afterhyperpolarization amplitude and decreasing the action potential threshold. None of these parameters were affected by 5-HT in adCINs. These results, together with our recent study of a third class of CINs, the aCINs whose axons ascend after crossing the midline (Zhong et al., 2006), suggest that dCINs and aCINs, but not adCINs, are excited by 5-HT and are rhythmically active during fictive locomotion. Thus, they may play important roles in the coordination of left-right movements during fictive locomotion.