Neuronal activity elicits metabolic and vascular responses, during which oxygen is first consumed and then supplied to the tissue via an increase in cerebral blood flow. Understanding the spatial and temporal dynamics of blood and tissue oxygen (To2 ) responses following neuronal activity is crucial for understanding the physiological basis of functional neuroimaging signals. However, our knowledge is limited because previous To2 measurements have been made at low temporal resolution (>100 ms). Here we recorded To2 at high temporal resolution (1 ms), simultaneously with co-localized field potentials, at several cortical depths from the whisker region of the somatosensory cortex in anaesthetized rats and mice. Stimulation of the whiskers produced rapid, laminar-specific changes in To2 . Positive To2 responses (i.e. increases) were observed in the superficial layers within 50 ms of stimulus onset, faster than previously reported. Negative To2 responses (i.e. decreases) were observed in the deeper layers, with maximal amplitude in layer IV, within 40 ms of stimulus onset. The amplitude of the negative, but not the positive, To2 response correlated with local field potential amplitude. Disruption of neurovascular coupling, via nitric oxide synthase inhibition, abolished positive To2 responses to whisker stimulation in the superficial layers and increased negative To2 responses in all layers. Our data show that To2 responses occur rapidly following neuronal activity and are laminar dependent.