The sustained entry of Ca(2+) into CD4(+)CD8(+) double-positive thymocytes is required for positive selection. Here we identified a voltage-gated Na(+) channel (VGSC) that was essential for positive selection of CD4(+) T cells. Pharmacological inhibition of VGSC activity inhibited the sustained Ca(2+) influx induced by positively selecting ligands and the in vitro positive selection of CD4(+) but not CD8(+) T cells. In vivo short hairpin RNA (shRNA)-mediated knockdown of the gene encoding a regulatory β-subunit of a VGSC specifically inhibited the positive selection of CD4(+) T cells. Ectopic expression of VGSC in peripheral AND CD4(+) T cells bestowed the ability to respond to a positively selecting ligand, which directly demonstrated that VGSC expression was responsible for the enhanced sensitivity. Thus, active VGSCs in thymocytes provide a mechanism by which a weak positive selection signal can induce the sustained Ca(2+) signals required for CD4(+) T cell development.