Sodium pentobarbital (Abbott Laboratories) was widely used as an intravenous (IV) anesthetic in rabbits, but it was removed from the market in 1995. Many commercially available alternatives, including Nembutal(r) (Abbott Laboratories), Euthanasia-5(r) (Veterinary Laboratories, Inc.), and Euthanasia-6(r) (Veterinary Laboratories, Inc.), contain sodium pentobarbital. However, all of these products produced an unacceptable level of hemolysis in antisera collected from anesthetized rabbits. The experimental design of this study included evaluating each component of these pentobarbital formulations to determine the exact cause of hemolysis. The goal of this study was to find an alternative formulation of sodium pentobarbital that did not produce hemolysis in rabbit sera obtained from terminal bleeds. Specific ingredients in all formulations included sterile water, alcohol, propylene glycol (PG), and sodium pentobarbital in various combinations and concentrations. Study animals were inoculated intravenously with either sterile water, 10% ethanol, 40% ethanol, 2.5% PG, 40% PG, or isotonic saline as a control. PG at 40% concentration produced the most severe hemolysis, followed by 40% ethanol and sterile water. Levels of hemolysis caused by 10% ethanol and 2.5% PG did not differ statistically from those produced by isotonic saline. Subsequently, our laboratory adopted a pentobarbital formulation consisting of sodium pentobarbital dissolved at a concentration of 50 mg/ml in 10% ethanol and sterile water. This formulation is non-hemolytic in rabbits.