Diastolic time fraction (DTF) has been recognized as an important determinant for subendocardial perfusion, but microsphere studies in which DTF 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 with fluorescent microspheres under full adenosine dilation. Perfusion pressure (P(c)) was defined as mean coronary arterial pressure minus minimal left ventricular pressure. Regional flow conductances (flow/P(c)) were as follows: for the subendocardium, C(endo) = -0.103 + 0.197 DTF + 0.00074 P(c) (P < 0.001); for the midmyocardium, conductance = -0.048 + 0.126 DTF + 0.00049 P(c) (P < 0.001); and for the subepicardium, C(epi) was not significant. C(endo)-DTF relations demonstrated a finite value for DTF at which flow is zero, 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 P(c): DTF1 = 0.78 - 0.003 P(c) (P < 0.01). When heart rate and P(c) were held constant and dobutamine was administered (5 goats), contractility doubled and DTF increased by 39%, resulting in an increase of C(endo) 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, C(endo) < C(epi) if P(c) is lower than approximately 75% of a normal aortic pressure and heart rate >80 beats/min.