Coronary blood vessels are compressed by the contracting myocardium. This leads to oscillations in flow in especially the subendocardium. We examined the effects of steady and oscillating flow on isolated, cannulated subendocardial and subepicardial porcine arterioles. Steady flow-induced dilation in both vessel types, up to 12.9+/-0.8% of the passive diameter in subendocardials and 9.6+/-1.4% in subepicardials at 40 dyne/cm2. Dilation was completely abolished after treatment with 10 micromol/L L-NNA. Sinusoidal modulation of steady flow at 1.5 Hz and 50% to 200% amplitude did not affect dilation. Oscillating flow without a net forward component with peak-peak shear values up to 100 dyne/cm2 caused no dilation at all in these vessels. However, in the presence of 100 U/mL superoxide dismutase (SOD), oscillating flow induced dilation up to 19.5+/-2.3% in subendocardial vessels and 11.5+/-4.3% in subepicardials. LNNA (10 micromol/L) blocked this dilation by approximately 50%. SOD did not affect the magnitude of steady flow-induced dilation, but the response time after onset of steady flow shortened from 23.4+/-1.5 to 14.3+/-2.1 seconds. Diphenyleneiodinium, an inhibitor of NAD(P)H oxidase, uncovered dilation to oscillating flow in subendocardial vessels up to 9.5+/-1.6%. Flow causes production of both NO and O2-. During steady flow, the bioavailability of NO is sufficient to cause vasodilation. During oscillating flow, NO is quenched by the O2-, suppressing vasodilation. Considering the pulsatile nature of subendocardial flow and the vulnerability of this layer, pharmacological manipulation of the balance between NO and O2- may improve subendocardial perfusion.