The partial pressure of oxygen (pO₂) and pH play critical roles in tumor biology and therapy. We report here the first combined, high-resolution (≤10 μm) measurements of interstitial pH and pO2 profiles between adjacent vessels in a human tumor xenograft, using fluorescence ratio imaging and phosphorescence quenching microscopy. We found (1) heterogeneity in shapes of pH and pO₂ profiles; (2) a discordant relation between local pH profiles and corresponding pO₂ profiles, yet a strong correlation between mean pH and pO₂ profiles; (3) no correlation between perivascular pH/pO₂ and nearest vessel blood flow; and (4) well-perfused tumor vessels that were hypoxic and, consequently, large hypoxic areas in the surrounding interstitium. Such multiparameter measurements of the in vivo microenvironment provide unique insights into biological processes in tumors and their response to treatment.