Functional magnetic resonance imaging (fMRI) is a powerful method for exploring emotional and cognitive brain responses in humans. However rodent fMRI has not previously been applied to the analysis of learned behaviour in awake animals, limiting its use as a translational tool. Here we have developed a novel paradigm for studying brain activation in awake rats responding to conditioned stimuli using fMRI. Using this method we show activation of the amygdala and related fear circuitry in response to a fear-conditioned stimulus and demonstrate that the magnitude of fear circuitry activation is increased following early life stress, a rodent model of affective disorders. This technique provides a new translatable method for testing environmental, genetic and pharmacological manipulations on emotional and cognitive processes in awake rodent models.