The K(v)4.2/4.3 channels are the primary subunits that contribute to the fast-inactivating, voltage-dependent transient outward K(+) current (I(to,fast)) in the heart. I(to,fast) is the critical determinant of the early repolarization of the cardiac action potential and plays an important role in the adaptive remodelling of cardiac myocytes, which usually causes cell volume changes, during myocardial ischaemia, hypertrophy and heart failure. It is not known, however, whether I(to,fast) is regulated by cell volume changes. In this study we investigated the molecular mechanism for cell volume regulation of I(to,fast) in native mouse left ventricular myocytes. Hyposmotic cell swelling caused a marked increase in densities of the peak I(to,fast) and a significant shortening in phase 1 repolarization of the action potential duration. The voltage-dependent gating properties of I(to,fast) were, however, not altered by changes in cell volume. In the presence of either protein kinase C (PKC) activator (12,13-dibutyrate) or phosphatase inhibitors (calyculin A and okadaic acid), hyposmotic cell swelling failed to further up-regulate I(to,fast). When expressed in NIH/3T3 cells, both K(v)4.2 and K(v)4.3 channels were also strongly regulated by cell volume in the same voltage-independent but PKC- and phosphatase-dependent manner as seen in I(to,fast) in the native cardiac myocytes. We conclude that K(v)4.2/4.3 channels in the heart are regulated by cell volume through a phosphorylation/dephosphorylation pathway mediated by PKC and serine/threonine phosphatase(s). These findings suggest a novel role of K(v)4.2/4.3 channels in the adaptive electrical and structural remodelling of cardiac myocytes in response to myocardial hypertrophy, ischaemia and reperfusion.