Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K+ (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K+ currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K+ current ( Ito) that activated at potentials positive to −40 mV and partially inactivated during depolarizing voltage steps. Ito was inhibited by the antiarrhythmic agent flecainide (100 μM) and BaCl2 (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K+ currents [ IK fast ( IKf) and IK slow ( IKs)] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited Ito but had no effect on IKf and IKs. Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 α-subunits but not Kv4.2 or Kv1.6 α-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca2+ mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.