The goal of this study was to determine whether inward Na+ or Ca2+ currents could be measured in cardiac microvascular endothelial cells (CMEC). CMEC were isolated from rat ventricular muscle and studied during days 1-4 in culture. Differential uptake of fluorescently labeled acetylated low-density lipoproteins (LDL) indicated that the primary culture contained > 90% CMEC. Membrane currents were measured with the use of the whole cell arrangement of the patch-clamp technique with a Cs+ internal solution to prevent contamination by outward K+ currents. Voltage steps positive to -30 mV resulted in the activation of a fast, inward Na+ current (INa). In 20 cells examined, the peak inward current measured at 0 mV was 2.1 pA/pF. The half-maximal voltage required for inactivation of INa was -45 mV, and the current recovered from inactivation with a time constant of 10 ms. Inward currents were eliminated by replacement of external sodium with N-methylglucamine and were blocked by both tetrodotoxin (TTX) (dissociation constant = 5 nM) and saxitoxin (50 nM). Stimulation of protein kinase C, through application of phorbol 12,13-dibutyrate, resulted in an increase in the amplitude of INa without any change in the voltage dependence of current activation. Thus the endothelium of cardiac microvessels may be unique in expressing voltage gated, TTX-sensitive Na+ channels.