To compare the feasibility of real-time myocardial contrast echocardiography (MCE) in rats with infusion and bolus administration of a second-generation ultrasound contrast agent BR1. B-mode real-time MCE was performed in 12 Sprague Dawley rats following the BR1 infusion or bolus injection. The myocardium signal intensity (SI) was plotted against time and was fitted to exponential functions. The plateau SI (A) and rate of SI increase (beta) for the infusion study and peak signal intensity (PSI) for the bolus study were obtained. (99m)Tc-Sestamibi and Evans blue were used to assess myocardial blood perfusion and to calculate the myocardium perfusion defect area ex vivo. High-quality real-time MCE images were successfully obtained using each method. At baseline, all LV segments showed even contrast distribution. Following left anterior descending coronary artery (LAD) ligation, significant perfusion defect was observed in LAD beds with a significantly decreased A* beta and PSI values compared with LCx beds (Infusion: A*beta (LAD): 5.42 +/- 1.57 dB, A*beta (LCx): 46.52 +/- 5.32 dB, p < 0.05; Bolus: PSI (LAD): 2.11 +/- 0.67 dB, PSI (LCx): 20.68 +/- 0.72 dB, p < 0.05), which was consistent with (99m)Tc-Sestamibi distribution findings. Myocardial perfusion defect areas, assessed by both methods, showed no differences and showed good correlation with Evans blue staining. ED frames were more favorable for imaging analysis. Both infusion and bolus administration of the contrast agent combined with real-time MCE technique can provide a reliable and noninvasive approach for myocardial perfusion assessment in rats and the infusion method was more suitable for quantitative analysis of myocardial blood flow.