Methotrexate (MTX) is a cytostatic agent used in adjuvant chemotherapy for treatment of breast cancer and is associated with cognitive impairment in a subgroup of patients. The aim of this paper is to test whether MTX can rapidly affect various brain structures resulting in decreased hippocampal cell proliferation and white matter damage. We also studied whether cell death occurs in the hippocampus following MTX. All these processes may contribute to the memory deficits reported in patients. The first study explored the effect of an intravenously injected high-dose MTX (250 mg/kg) on hippocampal cell proliferation, white matter, and cell death. Proliferation was not significantly decreased 1 day after administration of MTX, although a high individual variation was seen. However, 7 days after MTX treatment hippocampal cell proliferation was significantly lower compared to control animals. White matter density was decreased in the lateral corpus callosum of animals treated with MTX, 1 day, 1 week, and 3 weeks after treatment. MTX did not induce hippocampal cell death at any of the time intervals after treatment. The second study examined the effect of MTX on memory by subjecting animals to a learning task directly followed with MTX treatment. In both learning tasks, memory was impaired in treated animals. In the Morris water maze, animals treated with MTX spent significantly less time in the correct quadrant compared to control animals during the probe trial which was performed 1 week after training and treatment. In contextual fear conditioning, animals treated with MTX showed significantly less freezing behavior compared to control animals, 4 weeks after training and treatment. These studies suggest that the negative effect of MTX on hippocampal cell proliferation and white matter density may be part of the mechanisms underlying the cognitive impairment observed as side effect after cytotoxic treatment in humans.