The trial-unique, delayed nonmatching-to-location (TUNL) task is a recently developed behavioral task that measures spatial working memory and a form of pattern separation in touchscreen-equipped operant conditioning chambers. Limited information exists regarding the neurotransmitters and neural substrates involved in the task. The present experiments tested the effects of systemic and intracranial injections of NMDA receptor antagonists on the TUNL task. After training, male Long Evans rats systemically injected with the competitive NMDA receptor antagonist CPP (10 mg/kg) had impaired accuracy regardless of the degree of stimuli separation or length of delay between the sample and test phases. Injections of Ro 25-6981 (6 or 10 mg/kg), an antagonist selective for GluN2B subunit-containing NMDA receptors, did not affect accuracy on the task. Direct infusion of the competitive NMDA receptor antagonist AP5 into mPFC or dmSTR reduced overall accuracy on the TUNL task. These results demonstrate that TUNL task performance depends on NMDA receptors within the mPFC and dmSTR.