Myocardial SPECT may be useful for assessment of the therapeutic effects and the mechanisms of cardiac regeneration medicine. We aimed to assess first the feasibility and the short-term safety of autologous bone marrow–derived mononuclear cell transplantation (BMCT) into the ischemic myocardium in patients who undergo off-pump coronary artery bypass surgery (OPCAB). In addition, we aimed to assess our hypothesis that the BMCT may help ameliorate myocardial perfusion in patients with ischemic heart disease (IHD) using myocardial perfusion scintigraphy. We performed BMCT in 10 patients with IHD during OPCAB. Cells for BMCT were collected by intraoperative bone marrow aspiration or by preoperative cellular apheresis after pretreatment with granulocyte colony-stimulating factor. After OPCAB was performed in all graftable ischemic areas, a total of 3.4 ± 1.2 × 10⁹ mononuclear cells, including 5.2 ± 1.6 × 10⁶ CD34-positive (CD34+) cells, were injected into ungraftable ischemic myocardial areas. Dipyridamole-stress and resting ^99mTc myocardial SPECT was performed before and 1 mo after the procedures. BMCT was performed safely in all patients. Compared with before treatment, myocardial ^99mTc tracer uptake on the dipyridamole-stress image increased similarly in BMCT- and OPCAB-treated areas, whereas tracer accumulation at rest did not change in all myocardial areas. The improvement of myocardial perfusion was not correlated with the total number of mononuclear cells transplanted. However, it was positively correlated with the number of transplanted CD34+ cells: ^99mTc tracer uptake after/before BMCT (ratio) = 1.091 × (CD34+ cell number [×10⁶])^0.074 (r² = 0.48, P < 0.05), although new development of coronary vessels was not documented cineangiographically. Myocardial histopathology in 2 of 3 autopsy cases revealed coronary angiogenesis in the areas corresponding to the sites of BMCT. The present study demonstrates the feasibility of BMCT combined with OPCAB. This therapy improves myocardial perfusion possibly via CD34-related development of coronary microvessels.