In this study we compare oxygen tension (PO2) histograms measured with O2 microelectrodes and a new optical PO2 measurement device, the OxyLite, in normal tissues (mouse spleen and thymus) and in tumors (R3230Ac in rats) (n = 5-6). The transient response to glucose infusion or 100% O2 breathing (hyperoxia) was also measured in tumors. PO2 histograms of spleen and thymus with the two devices were not different. The OxyLite tumor PO2 histogram, however, was left-shifted compared with the microelectrode (median PO2 1.0 vs. 4.0 mmHg, P = 0.016). Both probes responded to acute hyperglycemia with a mean increase of 3-6 mmHg, but the microelectrode change was not significant. The OxyLite consistently recorded large PO2 increases (approximately 28 mmHg) with hyperoxia, whereas the microelectrode response was variable. The OxyLite averages PO2 over an area that contains interstitial and vascular components, whereas the microelectrode measures a more local PO2. This study demonstrates the importance of considering the features of the measurement device when studying tissues with heterogeneous PO2 distributions (e.g., tumors).