Differential classical conditioning of the gill-withdrawal response (GWR) in Aplysia can be elicited by training in which a conditioned stimulus (CS) delivered to one side of the siphon (the CS+) is paired with a noxious unconditioned stimulus (US; tail shock), while a second conditioned stimulus (the CS-), delivered to a different siphon site, is unpaired with the US. NMDA receptor (NMDAR) activation has been shown previously to be critical for nondifferential classical conditioning in Aplysia. Here, we used a semi-intact preparation to test whether differential classical conditioning of the GWR also depends on activation of NMDARs. Differential training produced conditioned enhancement of the reflexive response to the CS+ and a reduction in the response to the CS-. Comparison of the results after differential training with those after training in which only the two CSs were presented (CS-alone experiments) indicated that the decrement in the response to CS- after differential training was not caused by habituation. Surprisingly, differential training in the NMDAR antagonist APV (DL-2-amino-5-phosphonovalerate) blocked not only the conditioned enhancement of the GWR, but also the conditioning-induced depression of the GWR. We suggest that differential conditioning involves an NMDAR-dependent, competitive interaction between the separate neural pathways activated by the CS+ and CS-.