We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V–VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles. J. Comp. Neurol. 519:3470–3487, 2011. © 2011 Wiley-Liss, Inc.