The role of the N-methyl-D-aspartate (NMDA) receptors in cocaine conditioning and sensitization of locomotor activity was studied in four groups of Sprague-Dawley rats. A sub-motoric dose of the NMDA antagonist MK-801 (0.1 mg/kg, i.p.) was employed using a novel dual-compartment Pavlovian drug conditioning paradigm. The animals were placed sequentially in two different test environments in which locomotor activity was monitored. In the first compartment, the animals always received a non-drug test for 20 min. Upon completion of this test, the animals received either saline, cocaine (10 mg/kg i.p.), MK-801 or MK-801 plus cocaine depending on group assignment and were then placed immediately into the second compartment and again tested for 20 min. A total of six non-drug and six drug tests were conducted every other day over a 12-day period. Across all drug/saline treatment and post-treatment tests for conditioning, there were no statistical differences in locomotor activity among the saline and drug treatment groups in the non-drug test environment. In the drug/saline associated environment, however, cocaine had a reliable stimulant effect on locomotion when administered alone or in combination with MK-801. Following a 1-day and again after 21-days of withdrawal, all animals were administered a non-drug test for conditioning in which no injections were administered. On both tests, all groups had equivalent activity levels in the non-drug environment. In the drug/saline environment, only the cocaine group of the three drug treatment groups exhibited conditioned hyperlocomotion. Importantly, MK-801 blocked conditioned hyperlocomotion in the combined cocaine+MK-801 group. MK-801 did not alter serum or brain cocaine concentration or the cocaine effects on dopamine metabolism in limbic brain tissue. The co-administration of MK-801 with cocaine, however, blocked the corticosterone release effect of cocaine. Thus, the NMDA receptor site appears critical for cocaine induced conditioning and for corticosterone release.