The mechanisms responsible for coronary pressure-flow autoregulation, a critical physiologic phenomenon that maintains coronary blood flow relatively constant in the presence of changes in perfusion pressure, remain poorly understood. This investigation tested the hypothesis that voltage-sensitive K⁺ (K_V) and Ca²⁺ (Ca_V1.2) channels play a critical role in coronary pressure-flow autoregulation in vivo. Experiments were performed in open-chest, anesthetized Ossabaw swine during step changes in coronary perfusion pressure (CPP) from 40 to 140 mmHg before and during inhibition of K_V channels with 4-aminopyridine (4AP, 0.3 mM, ic) or Ca_V1.2 channels with diltiazem (10 μg/min, ic). 4AP significantly decreased vasodilatory responses to H₂O₂ (0.3–10 μM, ic) and coronary flow at CPPs = 60–140 mmHg. This decrease in coronary flow was associated with diminished ventricular contractile function (dP/dT) and myocardial oxygen consumption. However, the overall sensitivity to changes in CPP from 60 to 100 mmHg (i.e. autoregulatory gain; Gc) was unaltered by 4-AP administration (Gc = 0.46 ± 0.11 control vs. 0.46 ± 0.06 4-AP). In contrast, inhibition of Ca_V1.2 channels progressively increased coronary blood flow at CPPs > 80 mmHg and substantially diminished coronary Gc to −0.20 ± 0.11 (P < 0.01), with no effect on contractile function or oxygen consumption. Taken together, these findings demonstrate that (1) K_V channels tonically contribute to the control of microvascular resistance over a wide range of CPPs, but do not contribute to coronary responses to changes in pressure; (2) progressive activation of Ca_V1.2 channels with increases in CPP represents a critical mechanism of coronary pressure-flow autoregulation.