Introduction: To date, investigation of coronary arteriole vasomotor activity has been limited to arterioles >30– 40 µm. Here, we introduce a new experimental model to allow for in situ microscopy of terminal coronary arterioles. Methods: Rat hearts were perfused in a closed loop system (priming volume 20 ml) which was placed on a computer-controlled microscope stage. FITC-dextran and tetrodotoxin (TTX, 50 µ M) were added. Tilting of the microscope by 90° allowed for visual access to the ventricular surface. Arterioles were identified by the flow direction of fluorescent beads (1 µm). Images were recorded on video tape, and arteriole diameters were measured offline. Stability of the preparation and maintenance of coronary flow reserve were analyzed. Responses of coronary flow and arteriole diameters to the vasodilators papaverine and Na-nitroprusside were recorded. Results: In TTX-arrested control hearts coronary flow and terminal arteriole diameters were stable for 2 h. Administration of papaverine and Na-nitroprusside increased coronary flow from 6.4 ± 0.7 to 13.3 ± 1.3 ml/min, decreasing coronary resistance by 52 ± 3%. Terminal coronary arteriole diameters increased from 12.0 ± 0.9 to 13.6 ± 1.0 µm, decreasing hindrance of this vessel segment by 45 ± 11%. Conclusion: Preservation of coronary terminal arteriolar tone and adequate responsiveness to vasodilators in the TTX-arrested isolated heart were demonstrated. Thus, this model may serve to complement our understanding of coronary microvascular control mechanisms by extending observations to the terminal arteriolar bed.