Accumulated evidence suggests that the heteromeric assembly of Kv4.2 and Kv4.3 alpha-subunits underlies the fast transient Kv current (I(to,f)) in rodent ventricles. Recent studies, however, demonstrated that the targeted deletion of Kv4.2 results in the complete elimination of I(to,f) in adult mouse ventricles, revealing an essential role for the Kv4.2 alpha-subunit in the generation of mouse ventricular I(to,f) channels. The present study was undertaken to investigate directly the functional role of Kv4.3 by examining the effects of the targeted disruption of the KCND3 (Kv4.3) locus. Mice lacking Kv4.3 (Kv4.3-/-) appear indistinguishable from wild-type control animals, and no structural or functional abnormalities were evident in Kv4.3-/- hearts. Voltage-clamp recordings revealed that functional I(to,f) channels are expressed in Kv4.3-/- ventricular myocytes, and that mean I(to,f) densities are similar to those recorded from wild-type cells. In addition, I(to,f) properties (inactivation rates, voltage dependences of inactivation and rates of recovery from inactivation) in Kv4.3-/- and wild-type mouse ventricular myocytes were indistinguishable. Quantitative RT-PCR and Western blot analyses did not reveal any measurable changes in the expression of Kv4.2 or the Kv channel interacting protein (KChIP2) in Kv4.3-/- ventricles. Taken together, the results presented here suggest that, in contrast with Kv4.2, Kv4.3 is not required for the generation of functional mouse ventricular I(to,f) channels.