A delayed-match-to-sample for spatial location task was used to measure spatial pattern separation and working memory. On each trial, an object covered a baited food well in one of 15 spatial locations along a row of food wells perpendicular to the start box. Once the rat exited the start box, displaced the object to receive a food reward and then returned to the startbox, the same food well was then quickly re-baited, an identical object was positioned to cover the food well and another identical object was positioned in a different location along the row of food wells covering a different unbaited food well. On the ensuing choice phase, the animal was allowed to choose between the two objects. The object that covered the same food well as the object in the sample phase was the correct choice and the second foil object was the incorrect choice. Five spatial separations (15-105 cm) were randomly used to separate the correct object from the foil object during the choice phase. Once a preoperative criterion was met, each rat received bilateral intracranial infusions of either ibotenic acid or the vehicle into the CA3 subregion of the dorsal hippocampus. Following surgery, rats with CA3 lesions were significantly impaired relative to controls across all spatial separations suggesting that CA3 lesions impaired working memory. Although the dorsal CA3 subregion of the hippocampus may play a role in pattern separation, the data suggest that this region is critically involved in spatial working memory.