We used a conditionally immortalized cell line US/VOT-36 derived from the mouse embryonic cochlea to search for subunits of the embryonic and neonatal hair cell potassium (K⁺) currents I_K,emb and I_K,neo. It was derived from the otocyst at embryonic day 10 (E10, as described for a similar cell line, Lawoko-Kerali et al. 2004) and is likely to form a multipotent cell line capable of differentiation into hair cell-like cells. Whole-cell patch clamp recordings from cells grown under differentiating conditions showed endogenously expressed, voltage-activated K⁺ currents. They shared characteristics of delayed-rectifier K⁺ currents recorded from embryonic hair cells of humanely killed mice between E16 and the day of birth (P0), namely acquisition of a 4-AP sensitive partially inactivating component and a shift to a more hyperpolarized and steeper voltage dependence of activation (Marcotti et al. 2003; Helyer et al. in press). In both VOT-36 and basal outer hair cells (OHCs) at E18, 4-AP application removed the inactivating component. The remaining current resembled currents recorded earlier in differentiation in VOT-36 and in basal E16 OHCs. RT-PCR showed the presence of mRNA for Kv1.3 and Kv2.1 subunits in cell line cDNA and in whole organ of Corti cDNA prepared from P3 mice. Neither subunit has previously been shown to be expressed in cells of the mammalian organ of Corti. The changes in electrophysiological properties described are consistent with the expression of these subunits in VOT-36 and OHCs. Using immunohistochemistry, we showed Kv2.1 was expressed specifically on the basolateral membrane of inner hair cells (IHCs), with less expression in outer hair cells (OHCs). It was also expressed in hair cells of the utricular macula. Kv1.3 was present in IHC, OHCs and vestibular hair cells but no other cells of the sensory epithelium and was distributed diffusely throughout the cells. These results show that conditionally immortalized cell lines derived from the early embryo can express functional K⁺ channels during conditional differentiation that may be candidates for subunits underlying the currents expressed in mammalian hair cells during development of the sensory epithelium. We speculate that Kv2.1 and Kv1.3 may be molecular correlates of components of I_K,emb and I_K,neo.