We examined the effects of acute injections of competitive N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) into the dorsal hippocampus on contextual fear conditioning and classical eyeblink conditioning in C57BL/6 mice. When injected 10 to 40 min before training, APV severely impaired contextual fear conditioning. Thus, APV injection under these conditions was sufficient to suppress hippocampal NMDA receptors. To investigate the role of hippocampal NMDA receptors on eyeblink conditioning, we carried out daily training of mice during 10-40 min after injection of APV. In the delay eyeblink conditioning, in which the unconditioned stimulus (US) is delayed and terminates simultaneously with the conditioned stimulus (CS), APV-injected mice acquired the conditioned responses (CRs) as well as artificial cerebrospinal fluid (aCSF)-injected control mice did. However, in the trace eyeblink conditioning, in which the CS and US were separated by a stimulus-free trace interval of 500 ms, APV-injected mice showed severe impairment in acquisition of the CR. There was no significant difference in pseudo-conditioning between APV- and aCSF-injected mice. These results provide evidence that the NMDA receptor in the dorsal hippocampus is critically involved in acquisition of the CR in long trace eyeblink conditioning.