Previously we found that 1,3-butanediol-treated mice live longer during hypoxia. We hypothesized that 1,3-butanediol could reduce the brain's accumulation of potentially cytotoxic lactate and/or elevate brain substrate availability (ketones or glucose) and thus maintain the brain's energy producing capability even during reduced oxygen availability. To test these hypotheses, whole brain metabolites from normoxic and hypoxic mice, pretreated with 1,3-butanediol or insulin, were compared to saline controls. During hypoxia both pretreated groups had lower brain lactate than controls. If lactate accumulation was the sole factor responsible for hypoxic tolerance, insulin should have increased brain lactate since insulin has been shown previously to reduce hypoxic tolerance. In normoxic mice the ratio of lactate to pyruvate and the level of malate and fumarate were not changed by 1,3-butanediol as is found with other agents known to protect the hypoxic animal. When substrate availability was directly elevated by beta-hydroxybutyrate and glucose administration hypoxic survival time increased thus implicating substrate availability as an important factor in hypoxic tolerance. We conclude that reduced brain lactate and augmented substrate availability both contribute to 1,3-butanediol-enhanced hypoxic tolerance in this animal model.