Single Na+ channel currents have been examined in isolated guinea pig ventricular myocytes using the patch-clamp technique. The effects of lidocaine, extracellular calcium [(Ca)o], and tetrodotoxin on patch Na+ channel availability were assessed using ensemble averages of Na+-channel openings during depolarizing test potential steps from 7 to 10 different patch-holding potentials in each cell-attached patch. In six control patches, the potential for 50% channel availability (Vh) was -15 mV (relative to an average resting membrane potential of -80 mV). Exposure of patches to either lidocaine or elevated (Ca)o produced the expected shifts in Vh [average -22 mV for lidocaine and +10 mV for 6 mM (Ca)o]. Exposure of patches to tetrodotoxin (0.5 microM or 1.0 microM) produced a dose-dependent hyperpolarizing shift of Vh (average -10 and -17 mV) compared with control patches. The hyperpolarizing shift by tetrodotoxin was observed with pulses applied at frequencies of 1.0 or 0.067 Hz. In agreement with earlier maximal upstroke velocity studies in the same preparation, we conclude that block of ventricular Na+ channels by tetrodotoxin exhibits genuine steady-state voltage dependence.