In vivo experiments were done in mice to study the influence of binding of cloxacillin and nafcillin to serum on their antibacterial effect. Both antibiotics had the same activity in vitro against the Staphylococcus aureus strain used in these experiments. Binding to murine serum, as measured by ultrafiltration, was about 80% for cloxacillin and 89%–94% for nafcillin. Corresponding values were found with ultracentrifugation. Total concentrations in serum of mice after im administration were the same for the two antibiotics after a dose of 20 µg/ g and higher for cloxacillin after 40 µg/g. Bacterial counts in kidneys of mice infected iv with S. aureus 421 remained significantly higher after treatment with cloxacillin than after treatment with nafcillin at three dosage levels, indicating a weaker antibacterial effect of cloxacillin. This contrasts with what would be expected on the basis of data on MIC, serum binding, and serum concentrations taken together. Therefore, it is concluded that these data cannot be considered adequate under all circumstances for the prediction of therapeutic efficacy.