Cellular excitability is an important physiological factor in maintaining normal cardiac activity. The present study was designed to investigate the ionic mechanism underlying different excitability in atrial and ventricular myocytes of guinea pig heart using a whole-cell patch configuration. We found that excitability is lower in ventricular myocytes than that in atrial myocytes. Although the density of voltage-gated fast Na(+) current (INa) was lower in ventricular myocytes, it would not correlate to the lower excitability since its availability was greater than that in atrial myocytes around threshold potential. Classical inward rectifier K(+) current (IK1) was greater in ventricular myocytes than that in atrial myocytes, which might contribute in part to the lower excitability. In addition, the transient outward K(+) current with inward rectification (Itoir) elicited by depolarization was greater in ventricular myocytes than that in atrial myocytes and might contribute to the lower excitability. In ventricular myocytes, Ba(2+) at 5 µmol/L significantly inhibited Itoir, enhanced excitability, and shifted the threshold potential of INa activation to more negative, and the effect was independent of affecting INa. Our results demonstrate the novel information that in addition to classical IK1, Itoir plays a major role in determining the distinctive excitability in guinea pig atrial and ventricular myocytes and maintaining cardiac excitability. More effort is required to investigate whether increase of Itoir would be protective via reducing excitability.