This study assessed in vivo temporal and spatial electrophysiological properties of murine hearts and the effect of manipulation of transmural action potential durations (APDs) on T wave morphology. Monophasic action potentials (MAPs) were acquired from multiple left ventricular sites. All MAPs exhibited a plateau phase, with a spike and dome appearance being present in epicardial recordings. Activation occurred from endocardial apex to epicardial apex and apex to base while repolarization occurred from base (shortest MAP90 95.4 ± 8.9 ms) to apex and epicardium to endocardium (longest MAP90 110.77 ± 10.6 ms). The peak of phase 0 of the epicardial base MAP correlated with the return to baseline of the initial and usually dominant waveform of the QRS and the onset of the second usually smaller wave, which clearly occurred in early repolarization, thus establishing where depolarization ended and repolarization began on the murine ECG. This second waveform was similar to the J wave seen in larger animals. Despite temporal and spatial electrophysiological similarities, a T wave is frequently not seen on a murine ECG. There are several determinants of T wave morphology including: transmural activation time, slope of phase 3 repolarization, differences in epicardial, endocardial and M cell APDs. Experimental manipulation of murine transmural gradients by shortening epicardial MAP90 to 84% of endocardial MAP90, the epicardial / endocardial ratio in larger mammals when a positive T wave is present, resulted in a positive murine T wave. Thus, manipulation of the transmural gradients such that they are similar to larger mammals can result in T waves with similar morphology.