In this article, we present a first-pass perfusion imaging protocol to determine quantitative regional perfusion values (in mL min−1 g−1) of the mouse myocardium. Perfusion was quantified using a Fermi-constrained deconvolution of the myocardial tissue response with the arterial input function. A dual-bolus approach was implemented. Experimental evidence is presented for the linearity of signal intensity in the left-ventricular lumen during the prebolus (r = 0.99, P < 0.001) and in the myocardium during the full-bolus injection (r = 0.99, P < 0.01) as function of Gd(DTPA)2− injection concentration used. The prebolus was used to reconstruct a nonsaturated arterial input function. Regional perfusion values proved repeatable in a cohort of nine healthy C57BL/6 mice. The perfusion values over two measurements with a 1-week interval were 7.3 ± 0.9 and 7.2 ± 0.6 mL min−1 g−1, respectively. No effects of time (P > 0.05) and myocardial region (P > 0.05) were observed. The between-session coefficient of variation was only 6%, whereas the inter-animal coefficient of variation was 11 and 8% for the separate experiments. We expect that the first-pass perfusion method here presented will be useful in preclinical studies of myocardial perfusion deficits and valuable to assess the impact of pro-angiogenic therapy after myocardial infarction. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.