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Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na+ current is supported by Nav1.2 and/or Nav1.3 α-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Navβ2.1 transcript was not detected, whereas the Nav β1.1 transcript was present. Even in the absence of Navβ2.1, α-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K+ currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K+ current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv α-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K+ current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na+ and K+ channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.