AbstractUnder Na+-free conditions, low-voltage–activated Ca2+currents in cardiomyocytes from various species have been described either as Ni2+-sensitive T-type Ca2+current (ICa(T)) or as tetrodotoxin (TTX)-sensitive Ca2+current (ICa(TTX)). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under whichICa(T)andICa(TTX)can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na+-free solutions. Whereas rat myocytes lackICa(T)and exhibitICa(TTX)only, guinea pig myocytes possess both of these low-voltage–activated Ca2+currents, which are separated pharmacologically by superfusion with TTX or Ni2+.ICa(T)andICa(TTX)were of similar amplitude but significantly differed in their electrophysiological properties:ICa(TTX)activated at more negative potentials than didICa(T), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster.ICa(TTX)but notICa(T)increased after membrane rupture (“run-up”). Isolation ofICa(TTX)by application of the bivalent cation Ni2+is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca2+channel blocker mibefradil (10 &mgr;mol/L) is a suitable tool for the study ofICa(TTX). However, mibefradil not only blockedICa(T)by 85±2% but also decreasedICa(TTX)by 48±8%. We conclude that under Na+-free conditionsICa(T)andICa(TTX)coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations ofICa(T)will have to consider the TTX-sensitive current component to avoid possible interference.