Many new semisynthetic penicillins have been produced since 1959 when Batchelor et al1 isolated 6-aminopenicillanic acid. Many of these agents resist degradation by penicillinase and, hence, are efficacious against penicillinase-producing staphylococci. At present, two semisynthetic penicillins, methicillin and oxacillin, are commercially available. The purpose of this paper is to compare these two antibiotics with two new semisynthetic penicillins, nafcillin and cloxacillin, in vitro and in vivo. For the first time, a cephalosporin C derivative, cephalothin, has become available on a commercial scale, and this compound has also been included in the study. Methods and Materials Methicillin and oxacillin were supplied by Bristol Laboratories, Syracuse, NY. Cephalothin was obtained from Eli Lilly and Co., Indianapolis, as compound 38253. Nafcillin was supplied by Wyeth Laboratories, Inc., Radnor, Pa, as Wy 3277. Cloxacillin was supplied by Ayerst, McKenna & Harrison, Ltd., Montreal, as Ay 6203. Beecham Research Laboratories also supplied oxacillin and