An improved assay for racemic atenolol (AT) concentrations in human plasma and urine is described using a high-performance liquid chromatographic method with fluorescence detection. The method has a sensitivity limit of 0.5 micrograms/L in plasma with acceptable within- and between-run reproducibilities, and demonstrated linearity at concentrations up to 2,000 micrograms/L. A pilot clinical evaluation of the assay was undertaken on 56 trough plasma specimens from 36 outpatients on established AT therapy. Atenolol concentrations in these patients showed large variations at all prescribed doses, including undetectable levels in four patients (revealing unsuspected noncompliance). Because of its sensitivity and applicability to urinary analysis, the method can be used for pharmacokinetic studies and, under certain circumstances, may be valuable in clinical therapeutic drug monitoring.