We explored the feasibility of imaging myocardial perfusion and of demonstrating the flow changes in response to reduction of cardiac work non-invasively in anesthetized mice using high spatial resolution, dedicated small-animal positron emission tomography (microPET). In 31 C57BL/6 mice anesthetized with pentobarbital or isoflurane, (13)N-ammonia was injected intravenously and images were recorded with microPET from 4 to 20 min. Fifteen mice (group 1) were studied consecutively at baseline (BL) and after reduction of heart rate (HR) with intraperitoneal injection of clonidine (CLN) to investigate effects of CLN-induced reduction of cardiac work on myocardial (13)N-ammonia uptake. Eight mice (group 2) were imaged repeatedly at BL and eight mice (group 3) twice after CLN to examine reproducibility. Total myocardial (13)N-ammonia accumulation was determined from the transaxial images and normalized for injected dose (%ID). HR was 412+/-97 beats/min at BL and 212+/-44 beats/min after CLN (P<0.0001). In group 1, the %ID significantly decreased from 1.50%+/-0.27% at BL to 1.29%+/-0.28% after CLN (P<0.0001). In groups 2 and 3, reproducibility of %ID was good (y=0.96x+0.105, SEE=0.212%, r(2)=0.749, P<0.0001). In conclusion, (13)N-ammonia microPET imaging demonstrated non-invasively a reduction of myocardial perfusion induced by clonidine in mice. We believe this study is of importance as the first report on myocardial perfusion imaging and flow validation in in vivo mouse hearts with a left ventricular size of only 5 mm using (13)N-ammonia and PET. MicroPET will aid in elucidating cardiac pathophysiology in transgenic mice and monitoring effects of gene therapies on myocardial perfusion.