The isolated turtle brain maintains intra- and extracellular concentrations of ascorbate when incubated in ascorbate-free physiological saline for as long as 24 h. After incubation for 1 h, total tissue content of ascorbate in the turtle cerebellum was the same as in unincubated controls. After 20-24 h, tissue ascorbate content remained at 65% of control levels, while extracellular ascorbate concentration, measured with carbon fiber voltammetric microelectrodes, was 56% of the initial value. For an intermediate incubation period of 6 h, reduced ascorbate content was maintained at about 80% of control levels, regardless of whether incubation was under normal conditions or in the absence of glucose or oxygen. By contrast, only 4% of the ascorbate content of guinea pig brain slices remained after a 6 h incubation. Maintenance of high levels of ascorbate by the anoxia-resistant turtle brain could be an important factor in the amelioration of oxidative injury in this tissue. Inclusion of ascorbate in media used for in vitro studies of mammalian brain tissue is recommended.