Several antibiotics, including cephalothin, cephalexin, tetracycline, penicillin G, chloramphenicol, and nitrofurantoin, have been found to bind in vitro to specific components in the l00,000-g supernatant of rat liver homogenates. The major binding component has been purified by chromatography on DEAE-Sephadex A-50 and by gel filtration on Sephadex G-100. This component is a protein with an approximate molecular weight of 45,000; incubation of the protein with a sulfhydryl reagent causes dissociation into subunits. These properties and amino acid analysis of the protein indicate that it is similar to another hepatic binding protein designated “ligandin.” The relative binding, determined in the presence of excess of each antibiotic, shows that cephalothin is bound to the greatest extent, followed by cephalexin, tetracycline, penicillin, and other antibiotics. Polymyxin B and erythromycin were not bound at all. It is postulated that an anionic or an electronegative constituent within the antibiotic structure is necessary for binding to this protein. Since rat albumin bound the antibiotics to approximately the same extent, it is suggested, but not established, that this protein may be of importance in the uptake and transport of certain antibiotics as well as other ligands.