The Otsuka Long-Evans Tokushima fatty rat is an animal model of Type 2 diabetes mellitus (DM), which is characterized by diastolic dysfunction associated with decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a). The aim of this study was to examine whether gene transfer of SERCA2a can influence coronary blood flow and cardiomyocyte diameter in this model. DM rats were injected with adenovirus carrying SERCA2a (DM+SERCA) or beta-galactosidase gene (DM+betaGal). Coronary blood flow was measured in cross-circulated excised hearts 3 days after infection. Although in all groups coronary blood flow remained unchanged even if left ventricular (LV) volume or intracoronary Ca(2+) infusion was increased, the DM+SERCA group showed a sustained increase in coronary blood flow compared with the other groups. This result suggests that the sustained high coronary blood flow is a specific response in SERCA2a-overexpressed hearts. Although the LV weight-to-body weight ratio (LV/BW) and cardiomyocyte diameter were higher in the DM and DM+betaGal groups than in the non-DM group, in the DM+SERCA group, these measurements were restored to non-DM size. The percentages of collagen area in the three DM groups was significantly higher than results shown in non-DM rats, and there were no significant differences in collagen area percentage among the three DM groups. These results suggest that a lowered LV/BW by SERCA2a overexpression is due mainly to reduced size of cardiomyocytes without any changes in collagen area percentage. In conclusion, in DM failing hearts, SERCA2a gene transfer can increase coronary blood flow and reduce cardiomyocyte size without reduction in collagen production.