We observed the comparative serum levels and mean peak serum antistaphylococcal activity in eight fasting adults who received 500 mg each of dicloxacillin, cloxacillin, oxacillin, and nafcillin. Dicloxacillin achieved higher and more prolonged serum levels and greater peak serum antistaphylococcal titers than the other drugs studied. The higher degree of protein binding of dicloxacillin was reflected in a greater disparity between the peak antistaphylococcal activity observed when dilutions were done in serum compared to broth. The lesser protein-bound penicillins showed less disparity, but this effect was offset by the higher serum levels obtained by dicloxacillin. The higher protein binding of dicloxacillin did not prevent its having equal or superior antistaphylococcal activity in serum when the drugs were given in equal doses.