Diastolic time fraction (DTF) has been recognized as an important determinant for subendocardial perfusion but microsphere studies in which diastolic time fraction was the independent variable are practically absent. In 21 anesthetized goats the left coronary main stem was artificially perfused at controlled pressure. DTF was varied by pacing the heart, vagus stimulation or administration of dobutamine. Regional coronary flow was measured using fluorescent microspheres under full adenosine dilation. Perfusion pressure (Pc) was defined as mean coronary arterial minus minimal left ventricular pressure. Regional flow conductance (C=flow/Pc) was as follows: For the subendocardium, Cendo = -0.103+0.197 DTF+0.00074Pc (p < 0.001) and for the midmyocardium, Cmid = -0.048+0.126 DTF+0.00049Pc (p < 0.001), while for the subepicardium, Cepi was not significant. Cendo - DTF relations demonstrated a finite value for DTF at which flow is zero (DTFzf) implying that, at physiological pressures, systolic subendocardial flow limitation extends into diastole. The DTF corresponding to an equal conductance in subendocardium and subepicardium (DTF1), was inversely related to Pc: DTF1= 0.78-0.003Pc (p<0.01). In case HR and Pc were constant and dobutamine was administered (5 goats), contractility (dPLv/dt max) doubled and DTF increased by 39%, resulting in an increase of subendocardial conductance of 40%. It is concluded that 1) DTF is a determinant of subendocardial perfusion, 2) systolic compression exerts a flow limiting effect into diastole, and 3) corresponding to clinical findings on inducible ischemia we predict that, under hyperemic conditions, Cendo 80 beats per minute.