A rapid, reliable procedure for the analysis of cloxacillin and/or nafcillin in 100 microliter of serum or plasma is described. Percentage analytical recovery of cloxacillin, nafcillin, and internal standard (5-(p-hydroxyphenyl)-5-phenylhydantoin) were 94, 91, and 85%, respectively. The between-day precision of the method at cloxacillin concentrations of 2, 8, and 20 mg/liter was 14.7, 11.6, and 10.3%, respectively. At identical serum concentrations, the values obtained for nafcillin were 15.3, 12.4, and 12.2%, respectively. The method will be used to study the pharmacokinetics of both drugs in patients with cystic fibrosis who are on long-term therapy. Preliminary data providing the i.v. half-life, clearance, and volume of distribution are presented for two normal individuals.