Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO2) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO2 can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO2 in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO2 in capillaries and by a biphasic PO2 response in the neuropil, consisting of an ‘initial dip’ and a rebound. 2PLM of PO2 opens new avenues for studies of brain metabolism and blood flow regulation.