OBJECTIVE It has been shown that oxygen gradients exist in R3230AC tumors grown in window chambers. The fascial surface is better oxygenated than the tumor surface. The purpose of the present study was to determine whether gradients exist for energy metabolites and other end points related to oxygen transport. METHODS Imaging bioluminescence was used to measure ATP, glucose, and lactate in cryosections of R3230AC tumors. Mean vessel density and hypoxic tissue fraction were assessed using immunohistochemistry. Tumor redox ratio was assessed by redox ratio scanning. RESULTS Lactate content and hypoxic fraction increased, whereas ATP, glucose, redox ratio, and vessel density decreased from the fascial to the tumor surface. CONCLUSIONS The data support a switch from aerobic to anaerobic metabolism concomitant with the PO2 gradient. The vascular hypoxia that exists in perfused vessels at the tumor surface leads to macroscopic tissue regions with restricted oxygen availability and altered metabolic status. Methods to reduce tumor hypoxia may have to take this into account if such gradients exist in human tumors. The results also have implications for hypoxia imaging, because macroscopic changes in PO2 (or related parameters) will be easier to see than PO2 gradients limited to the diffusion distance of oxygen.