NMDA receptors (NMDARs) contribute to phasic transmission and synaptic plasticity and are thought to be important for learning. To better understand where NMDAR signaling is necessary for learning, we combined viral genetic strategies with genetic mouse models to investigate the contribution of NMDARs in the dopamine system to appetitive Pavlovian conditioning. NMDAR signaling in dopamine neurons was not required for Pavlovian conditioning; however, NMDARs in D(1) dopamine receptor (D(1)R)-expressing medium spiny neurons (MSNs), which receive input from dopamine neurons, were critical for this type of learning. NMDAR signaling was also required in brain regions that project to dopamine neurons, because removing NMDARs from afferent neurons to the ventral tegmental area (VTA) also prevented learning. This effect was likely attributable to loss of NMDAR signaling in the neurons of the prefrontal cortex (PFC), because learning could be restored in these animals by rescuing NMDAR expression in the PFC. Moreover, removing NMDARs exclusively from the PFC also prevented learning. Our findings suggest that NMDARs in neurons that project to and receive projections from the VTA are necessary for Pavlovian conditioning and specifically implicate the PFC and D(1)R-expressing MSNs in associative learning.