Using a group of penicillins all belonging to the same chemical class, antibacterial activity against Staphylococcus aureuswas determinedin vitro and also in vivo by use of an intraperitoneal infection in mice. The compounds all showed essentially the same level of activity in vitro but differed markedly in their activity in vivo. This activity in vivo could be correlated directly with the extent of binding in mouse serum.