Voltage-gated K+ (Kv) channel accessory (beta) subunits associate with pore-forming Kv alpha subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv beta subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kvbeta1 gene (Kvbeta1-/-), the studies here were undertaken to explore directly the role of Kvbeta1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kvbeta1-/- and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean+/-SEM I(to,f) density is significantly (P< or =0.01) lower in Kvbeta1-/- (21.0+/-0.9 pA/pF; n=68), than in WT (25.3+/-1.4 pA/pF; n=42), LVA myocytes, and that mean+/-SEM I(K,slow) density is significantly (P< or =0.01) higher in Kvbeta1-/- (19.1+/-0.9 pA/pF; n=68), compared with WT (15.9+/-0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of I(K,slow), I(K,slow2,) is selectively increased in Kvbeta1-/- LVA myocytes. In parallel with the alterations in I(to,f) and I(K,slow2) densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kvbeta1-/- ventricles. Taken together, these results demonstrate that Kvbeta1 differentially regulates the functional cell surface expression of myocardial I(to,f) and I(K,slow2) channels.