1,3-Butanediol (BD) treatment was previously shown to produce a dose-related increase of the plasma levels of D-beta-hydroxybutyrate (BHB) and to protect brain tissue against hypoxia and ischemia. The purpose of this study was to test whether BD-induced hyperketonemia was associated with changes in brain extracellular and tissue concentrations of BHB. Changes in extracellular levels of BHB were continuously monitored in anesthetized rats before and after intraperitoneal injection of BD (25 mmol/kg), using intracerebral microdialysis coupled to online analysis of BHB in the dialysate. Cortical tissue concentrations of BHB were determined in control and BD-treated rats (25 and 50 mmol/kg, i.p.) after freezing of the brain in situ. Butanediol produced a rapid increase in dialysate levels of BHB, with a linear relationship between dialysate and plasma BHB concentrations (r = 0.81, p < 0.001). In contrast, and although brain tissue levels of BHB were markedly increased after BD treatment, they were not related to the plasma concentration of BHB. Our results suggest that BHB produced from BD did not accumulate in brain and that BD protects against hypoxia or ischemia by increasing brain BHB availability.