An object location test (OLT) has been developed to test spatial memory in rats. The test is based on the spontaneous tendency of rodents, previously exposed to two identical objects, to later explore one of the objects — replaced in a novel location — for a longer time than they explore the non-displaced object. In this study, we established the OLT in mice and investigated its characteristics with behavioral and pharmacological analysis. Mice discriminated the object in the novel location when the test trial was conducted ≤2 h after the acquisition trial. The cognitive ability was influenced neither by a change in the arrangement of the objects in the experimental apparatus, nor by change in the entry position of the mice. Object location memory was disrupted with change in the relative position of the objects to extra-field cues, or under conditions of deprivation of extra-field cues, suggesting that discrimination of the displaced object reflects spatial memory. A muscarinic cholinergic receptor antagonist (scopolamine, 1 mg/kg) impaired object location memory, while an acetylcholine esterase inhibitor (donepezil, 3 mg/kg) increased ability to maintain object location memory. In addition, aged mice showed poorer cognitive performance than young mice on the OLT. These findings indicate that the OLT can be used to assess spatial memory in mice, as well as in rats. The object location memory in mice was sensitive to pharmacological manipulation with cholinergic agents and to aging and could be used to identify agents affecting spatial memory.