Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A1 receptor (A1AR) knockout mice (A1KO) and their wild type controls (A1WT) were employed in this investigation. The heart and aortic arch were carefully removed and retro-infused with enzyme solution (1 mg/ml collagenase Type I, 0.5 mg/ml soya trypsin inhibitor, 3% bovine serum albumin, and 2% antibiotic) through aortic arch. The efflux was collected at 30, 60, and 90 min intervals. Collected cells were centrifuged and the pellets mixed with media (M199/F12 with 10% FBS with 2% antibiotics for endothelial cells; advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax and 2% antibiotic for smooth muscle cells) and plated. Endothelial cells were then characterized by a cobblestone appearance and positive Dil-Ac-LDL staining. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was about 91%. The western blot analysis showed expression of smoothelin in the cells from 3^rd, 7^th, and 11^th passages in both A1WT and A1KO. Further, the A1AR was characterized by western blot analysis using an A1AR specific antibody. To our knowledge, this is the first time that smooth muscle cells from the mouse coronary system have been isolated and successfully characterized.