Background Right ventricular (RV) pacing impairs left ventricular (LV) mechanics. In contrast, biventricular (BIV) pacing can improve LV efficiency in the setting of electromechanical dysfunction, thereby constituting the rationale for cardiac resynchronization therapy (CRT). Critical issues such as correct patient selection and the molecular effects of CRT are still elusive, and CRT models currently rely on large animals, which are expensive and cannot be manipulated genetically. Using a methodology for efficient implantation of cardiac electrodes in rodents, we explored the effect of pacing site on LV synchronization in rats. Methods Two miniature bipolar hook electrodes were implanted in vivo in the rat heart as follows: Group A (n = 6) right atrium (RA) and apical RV sites; group B (n = 7) RV and posterobasal LV sites; and group C (n = 7) similar implantation as in group B in combination with left coronary artery ligation. Electrodes were exteriorized through the back of the animal. Following postoperative recovery, two-dimensional transthoracic echocardiography was performed during pacing through the different electrodes. Segmental systolic circumferential strain (CS) analysis was used to evaluate LV dyssynchrony. Results In group A, RA pacing compared to sinus rhythm had no effect on LV synchrony as measured by standard deviation (SD) of segmental time to peak CS, SD of time to peak CS, and average delay between opposing ventricular segments. In contrast, RV pacing induced marked dyssynchrony ( P Conclusions In rats, LV and BIV pacing created favorable LV mechanics compared to RV pacing. These effects of pacing were less marked in the presence of extensive ischemic cardiomyopathy. Thus, rodent pacing appears to mimic important features seen in large mammals and may become a simple and reliable tool for pacing-related studies.