The transient outward current (I_to) is a major repolarizing current in the heart. Marked reduction of I_to density occurs in heart failure, and is accompanied by significant action potential duration (APD) prolongation. To understand the species-dependent role of I_to in regulating the ventricular action potential morphology and duration, we introduced simulated I_to conductance in guinea pig and canine endocardial ventricular myocytes using the dynamic clamp technique and perforated patch clamp recordings. The effects of simulated I_to in both types of cells were complex and bi-phasic, separated by a clear density threshold of about 40 pA/pF. Below this threshold, simulated I_to resulted in a distinct phase 1 notch, and had little effect on or moderately prolonged the APD by up to ~20%. I_to above the threshold resulted in all-or-none repolarization and precipitously reduced the APD. Qualitatively, these results confirmed our previous studies in canine ventricular cells using the whole-cell recordings. Further, the effects of simulated I_to are not linked to the particular mathematical formulations of the I_to model, or a specific stimulation rate. We conclude that, contrary to results of previous gene transfer studies, the response of guinea pig ventricular myocytes to a fully-inactivating I_to is similar to that of canine ventricular cells, and that in large animals such as dogs, I_to does not play a major role in setting the APD of left ventricular myocytes.