1. The aim of the present study was to examine the acute and chronic effects of adenoviral gene transfer on cardiac function in terms of left ventricular (LV) mechanoenergetic function. Recombinant adenoviral vector carrying beta-galactosidase and green fluorescent protein genes (Ad.betagal-GFP) was used. Cardiac function was examined in cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relationships (ESPVR/EDPVR), systolic pressure-volume area (PVA), LV relaxation rate, equivalent maximal elastance at mid-range LV volume (eE(max) at mLVV), coronary blood flow, coronary vascular resistance and myocardial oxygen consumption (VO(2)) were also measured. 2. To examine the ex vivo acute effects of the adenoviral vector, data were obtained before and 30-90 min after intracoronary infusion of Ad.betagal-GFP in the excised, cross-circulated hearts that underwent serotonin pretreatment. To examine the in vivo chronic effects of adenoviral gene transfer, normal rat hearts received Ad.betagal-GFP or saline by a catheter-based technique and data were obtained 3 days after the injection of Ad.betagal-GFP or saline. 3. The ESPVR, EDPVR, LV relaxation rate, eE(max) at mLVV, coronary blood flow and coronary vascular resistance remained unchanged in Ad.betagal-GFP-transfected hearts in both ex vivo acute and in vivo chronic experiments. Moreover, the ex vivo and in vivo transfection caused no change in the slope and VO(2) intercept of the VO(2)-PVA relationship, VO(2) for basal metabolism and for Ca(2+) handling in excitation-contraction coupling and O(2) costs of LV contractility. 4. These results indicate that adenoviral gene transfer has neither acute nor chronic toxic effects on LV mechanical and energetic function. A special combination of in vivo adenoviral gene transfer and a cross-circulation experimental system may provide a useful novel strategy to explore the functional and mechanoenergetic role of specifically targeted genes in the diseased heart.