Background Time-intensity curves derived from microbubble destruction/refilling sequences and recorded using myocardial contrast echocardiography (MCE) can provide parameters that correlate with coronary blood flow. The response of these parameters to adenosine vasodilatation correlates with coronary flow reserve (CFR) measured by fluorescent microsphere techniques (FMT). Currently, no data exist regarding the effect of physiological variables, such as hypoxia, on the determination of CFR by MCE. The purpose of this study was to define the effects of decreases in blood partial pressure of oxygen (PO2) on CFR as measured by MCE. Methods Studies were performed in 9 closed chest swine. Low-energy, real-time MCE was performed with commercial instruments in short axis view at papillary muscle level while infusing BR1 at 30 ml/h. High-energy ultrasound bursts (referred to as FLASH frames) destroyed the bubbles every 15 cardiac cycles, and resultant time-intensity curves derived from these sequences were fitted to the exponential function y = A (1-e-bt) +c, from which the rate of signal rise (b) was obtained. CFR was calculated as the ratio of b values after adenosine infusion to baseline and was obtained during the control period and after decreasing blood PO2 by giving nitrogen via a respirator to create artificial hypoxic conditions. CFR was independently determined by FMT. Results Nitrogen led to significant decreases in mean PO2, from (120.6±18.9) mmHg to (51.8±15.9) mmHg (P<0.01). Adenosine produced a similar increase in CFR (2.5 fold vs 3.1 fold) as assessed by MCE and FMT during the control period. The decrease in PO2 post nitrogen resulted in a slight increase in values at rest: 0.46±0.15 to 0.53±0.18 for b and (1.39±0.66) ml·min-1·g-1 to (1.72±0.30) ml·min-1·g-1 for myocardial blood flow (MBF) (both P<0.05). In addition, values decreased in response to adenosine using both techniques: 1.05±0.35 to 0.82±0.27 for b and (4.30±3.16) ml·min-1·g-1 to (3.93±1.27) ml·min-1·g-1 for MBF (both P<0.05). Thus, CFR was markedly reduced under hypoxic conditions, to 1.4 by MCE (P<0.05 compared with the baseline), and to 2.5 by FMT (P>0.05 compared with the baseline). Conclusions CFR values diminish under hypoxic conditions according to both MCE and FMT. The reductions in CFR involve both an increase in resting values and a decrease in post adenosine measurements, as determined by both techniques. The reduction in CFR under hypoxia is slightly greater using MCE than using FMT. Physiological variables, such as hypoxia, must be taken into consideration when assessing CFR by MCE.