An assay for the quantitative estimation of 4-aminopyridine in biological fluids has been developed using 2-aminopyridine as internal standard and ion-pair reversed-phase (C18) high-performance liquid chromatography with detection at 263 nm. A 7.5% solution o acetonitrile in water containing tetrabutylammonium iodide and sodium heptanesulfonate buffered at pH 3.0 provided excellent separation of the analytes from each other and from an interfering peak that was occasionally observed in the outdated human sera used in these studies. Sensitivity, specificity, precision, accuracy and reproducibility all were judged sufficient for the routine use of this assay for pharmacokinetic and pharmacodynamic studies.