Binding of pentobarbital (sodium 5-ethyl 5-(1-methylbutyl)barbiturate) with plasma protein of male and female rats was studied in vivo using the modified Rehberg's method. The female rat had more extensive binding capacity than the male rat, but the fraction of drug bound was similar in both sexes. As a result of castration, the degree of sex difference in the binding capacity lowered. When SKF -525 A, the inhibitor of metabolism of pentobarbital, was preadministered to the rat, blood level and amount bound of pentobarbital remained at the highest level, but fraction of drug bound was constant. The extent of binding was lowered in the rat pretreated with ether.