Cardiac resynchronization therapy (CRT) can improve global left ventricular (LV) function. However, limited data are available on regional LV contractility at rest and during exercise. The aim of the present study was to prospectively investigate the effects of CRT on regional LV ejection fraction (EF), global LVEF, and dyssynchrony, during rest and exercise, using radionuclide angiography. A total of 32 consecutive patients with heart failure and nonischemic cardiomyopathy underwent technetium-99m radionuclide angiography with bicycle exercise immediately after CRT implantation (during spontaneous rhythm and after CRT activation) and 3 months later. The regional EF was assessed in the interventricular septum and the lateral wall (LW). Intraventricular dyssynchrony was evaluated using Fourier phase analysis. During spontaneous rhythm, the EF was severely depressed in the septum compared to in the LW. CRT improved septal EF at rest and during exercise both at baseline (p <0.001) and after 3 months (p <0.05). The basal LW EF decreased during CRT (p <0.05, both at rest and during exercise). LV dyssynchrony decreased both at baseline and during follow-up, and the global LVEF showed improvement only at 3 months (p <0.001). In conclusion, in patients with nonischemic cardiomyopathy, CRT affects regional LV function by increasing the septal EF and reducing LW contractility, both at rest and during exercise. This was associated with an improvement in global LVEF and dyssynchrony.