Salmeterol (as the hydroxynaphthoate) is the first of a new class of long-acting beta-adrenergic receptor agonists with both bronchodilator and anti-inflammatory actions. A sensitive, accurate, and precise high-performance liquid chromatographic method for the determination of salmeterol in rat and dog plasma is described. Samples are prepared by solid-phase extraction and, after chromatography of the extracts on a reversed-phase styrene/divinylbenzene analytical column, salmeterol is detected by fluorescence monitoring (excitation wavelength, 230 nm; emission wavelength, 305 nm). The method is sensitive to 1 ng/mL, at which concentration the coefficient of variation was 16.3% in a single assay run. Repeated analyses of quality control samples, nominally at 2 ng/mL, were carried out over a number of assay runs with a coefficient of variation of 10.4%. The method is specific for salmeterol with respect to endogenous plasma components and identified metabolites. The assay was applied to the analysis of salmeterol in plasma of rats and dogs from pharmacokinetic studies.