The neural substrate of cognitive reappraisal has been well-mapped. Individuals who successfully downregulate negative affect (NA) by reshaping their thoughts about a potentially emotional situation show augmented activity in the prefrontal cortex (PFC), with attenuated activity in the amygdala. We performed functional neuroimaging with experience sampling to determine whether individual differences in brain activation correspond to differences in real-life NA. While being scanned, 69 female students (aged 18–25 years) were asked to perform a cognitive reappraisal task. In addition, repeated assessments (5/day, 14 days) of affect and minor events in real-life were conducted. Individual t-maps were created for an instructed downregulation contrast (downregulate negative–attend negative) and an uninstructed regulation contrast (attend negative–attend neutral). Mean beta values were extracted from a priori defined regions of interest in the bilateral amygdala and PFC and were correlated with three daily life NA measures: baseline (mean) NA, NA variability, and NA reactivity to negative events. Only one out of twelve correlations for the amygdalae was nominally significant, which did not survive correction for multiple comparisons. PFC activation in the instructed and uninstructed regulation contrasts explained approximately 10% of the variance in NA reactivity; stronger recruitment during the attend-negative condition was correlated with lower reactivity levels. The degree to which individuals spontaneously engage frontal clusters may be a critical aspect of real-life emotional reactivity. The findings of this study provide a partial external validation of the cognitive reappraisal task, suggesting that frontal brain activation during implicit task conditions may have the strongest connection with real-life behaviors.