Object location task (OLT) has been used as a model of hippocampal-dependent memory. Despite this application, there is neither a consistent pharmacological validation of NMDA receptor modulation nor an evaluation of hippocampal participation in mice. In the OLT, mice were placed in the open field with two identical objects for 3 min and, after a delay of 30, 90, 180 or 360 min, one object was moved to a new location and the time spent exploring the objects in new, (novel) and old (familiar) locations was recorded. Our results showed that the mice were able to discriminate object location when tested either 90 or 180 min after training. Intraperitoneal administration of MK801 (NMDA receptors antagonist) or scopolamine (mACh antagonist) induced amnesic effects. On the other hand, D-cycloserine (NMDA agonist) or tacrine (cholinesterase inhibitor) were able to improve memory in the mice tested. In addition, lidocaine infusion in the hippocampal CA1 region 10 min before training blocked object location memory. In short, this work indicates that OLT is susceptible to modulation of NMDA receptors, cholinergic neurotransmission and it is the first to characterize the participation of the hippocampal CA1 region, in this task.