Male rats were infused i.v. with hexobarbital to obtain a burst suppression of 1 s or more in the EEG (SS). At SS the rats were killed and the concentration of hexobarbital was determined by HPLC in three parts of the brain. Acute tolerance (induced by a 1-h exposure at the SS level) was recorded as an approximately 20% increase in brain concentrations of hexobarbital at the last SS during the exposure when compared with concentrations recorded at the first SS in the controls. Increased brain concentrations (approximately 8%) at SS were recorded 24 h after induction of acute tolerance. After 48 h the increase was uncertain. Thus, acute tolerance to hexobarbital could have cumulative properties if new exposures are imposed after 24 h.