We used Pavlovian counterconditioning in rats to identify the neural mechanisms for appetitive-aversive motivational interactions. In Stage I, rats were trained on conditioned stimulus (CS)-food (unconditioned stimulus [US]) pairings. In Stage II, this appetitive CS was transformed into a fear CS via pairings with footshock. The development of fear responses was retarded in rats that had received Stage I appetitive training. This counterconditioning was associated with increased levels of phosphorylated mitogen activated protein kinase immunoreactivity (pMAPK-IR) in several brain regions, including midline thalamus, rostral agranular insular cortex (RAIC), lateral amygdala, and nucleus accumbens core and shell, but decreased expression in the ventrolateral quadrant of the midbrain periaqueductal gray. These brain regions showing differential pMAPK-IR have previously been identified as part of the fear prediction error circuit. We then examined the causal role of RAIC MAPK in fear learning and showed that Stage II fear learning was prevented by RAIC infusions of the MEK inhibitor PD098059 (0.5 microgram/hemisphere). Taken together, these results show that there are opponent interactions between the appetitive and aversive motivational systems during fear learning and that the transformation of a reward CS into a fear CS is linked to heightened activity in the fear prediction error circuit.