The Cai(2+)-insensitive transient outward current, ilo was studied at 20-24 degrees C in rat ventricular myocytes with the whole cell recording patch-clamp technique. The current was recorded before and after replacement of chloride by methanesulfonate or aspartate or in the absence and the presence of chloride channel blockers, SITS or 9-anthracene carboxylic acid. In control conditions (in the presence of external divalent cations, Ca2+ and Cd2+, Cd2+ being used to suppress Ca2+ current), ilo inactivation was composed of a fast and a slow component. When methanesulfonate was substituted for external Cl-, the peak current decreased to a variable extent, but the inactivation of the remaining current was still composed of a fast and a slow component. In contrast, the inactivation of the difference current was well fitted by a single exponential. The time to peak of the difference current was shorter than that of the current recorded either in the absence or the presence of methanesulfonate. Both activation- and steady-state inactivation-voltage curves were either unchanged (n = 4) or shifted by a few mV (5.5 mV, n = 14) towards positive potentials when methanesulfonate was substituted for Cl-. The current remaining in methanesulfonate reversed at potentials closed to EK. The difference current was composed of a peak and a steady-state component. The peak was suppressed by 4-aminopyridine whereas the steady-state component was not. The peak was also suppressed when pipette solution contained Cs+ instead of K+ but was still present when the Hepes concentration in both external and pipette media was increased 5-fold (50 mM vs. 10 mM). When aspartate was substituted for Cl- or when 2 mM SITS was added to the external solution (in the absence of Ca2+ and Cd2+ because aspartate is known to chelate Ca2+ ions and possibly other divalent cations), ilo was reduced to a similar extent in the two cases and the difference current was composed of a peak (inactivation fitted by a single exponential) and a steady-state component. The SITS-sensitive transient current reversed at a potential close to ECl. When 5 mM 9-anthracene carboxylic acid was added to external solution (in the presence of Ca2+ and Cd2+), the peak of the difference current was similar to that observed when Cl- was substituted by methanesulfonate. The difference current resulting from the substitution of methanesulfonate for chloride was not changed when the pipette solution contained either 50 mM EGTA (instead of 5 mM) or 10 mM EGTA and 10 mM BAPTA. The nature of Cs(+)- and 4-aminopyridine-sensitive transient outward current suppressed by chloride ion substitutes or chloride channel blockers is discussed.