Real-time in vivo oxygen amperometry, a technique that allows measurement of regional brain tissue oxygen (O2) has been previously shown to bear relationship to the BOLD signal measured with functional magnetic resonance imaging (fMRI) protocols. In the present study, O2 amperometry was applied to the study of reward processing in the rat nucleus accumbens to validate the technique with a behavioural process known to cause robust signals in human neuroimaging studies. After acquisition of a cued-lever pressing task a robust increase in O2 tissue levels was observed in the nucleus accumbens specifically following a correct lever press to the rewarded cue. This O2 signal was modulated by cue reversal but not lever reversal, by differences in reward magnitudes and by the motivational state of the animal consistent with previous reports of the role of the nucleus accumbens in both the anticipation and representation of reward value. Moreover, this modulation by reward value was related more to the expected incentive value rather than the hedonic value of reward, also consistent with previous reports of accumbens coding of “wanting” of reward. Altogether, these results show striking similarities to those obtained in human fMRI studies suggesting the use of oxygen amperometry as a valid surrogate for fMRI in animals performing cognitive tasks, and a powerful approach to bridge between different techniques of measurement of brain function.