A method is developed for the specific and sensitive determination of cethromycin concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC), using a high-performance liquid chromatographic-tandem mass spectrometry (MS) method. The mobile phase consists of 50% acetonitrile-0.05% acetic acid-5mM ammonium acetate; the column used is a C(8) reversed-phase stationary phase. The preparation of samples requires a solvent extraction step. The retention times for cethromycin and the internal standard are approximately 2.0 and 2.7 min, respectively, with a total run time of 3.5 min. Detection is carried out using electrospray MS in a multiple reaction monitor mode. The detection limits for cethromycin are 1 ng/mL for plasma and 0.2 ng/mL for BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of cethromycin in human subjects.