The acute effects of pentobarbital were measured by the times required for the loss of righting reflex (overturn point) and the pentobarbital brain concentration associated with the overturn. The test consisted of immersing the fish in a 0.3 mg/ml sodium pentobarbital in 0.1 M Tris buffer challenge solution until the overturn point was reached. To examine the development of tolerance the fish were pre-exposed to 0.1 M Tris buffer solutions containing 0.0, 0.010, 0.015 and 0.025 mg/ml of sodium pentobarbital for 6, 24 or 48 hr at which time the overturn times and the pentobarbital brain concentrations at overturn in the challenge solution were determined. The mean pentobarbital content in the brain at overturn of fish pre-exposed to 0.015 or 0.025 mg/ml solution was significantly higher (P less than .01) than in control fish. The loss of tolerance was determined at 3 hr after termination of the pre-exposure of the fish to the various pentobarbital solutions; tolerance was measured only in the group of animals pre-exposed to the 0.025 solution by the significant increase (P less than .01) in the pentobarbital brain levels over control fish. The equilibration curve for fish swimming in 0.025 mg/ml of sodium pentobarbital was determined for 48 hr. A steady state was attained within 6 hr with brain levels that reached approximately 80% the concentration of the external solution.