Ronactolol [(+/-)-4'-[2-hydroxy-3-(isopropylamino)propoxy]-p-anisanilide], a new aminopropanol derivative showing beta-adrenoreceptor blocking activity, was administered orally as capsules to healthy humans at three single doses (30, 60, and 120 mg). Two HPLC methods were developed separately for determination of drug levels in urine and plasma. For plasma samples, after addition of internal standard (IS), a single-step extraction of alkalinized plasma was performed with methylene chloride. The organic layer was evaporated to dryness under reduced pressure, and the residue was taken up and chromatographed on a microbore silica column. Ronactolol and IS were detected by a UV detector at a wavelength of 278 nm. Excellent linearity was observed between the peak height ratios (ronactolol:IS) and concentrations in plasma. The lowest limit of detection (signal:noise, 3:1) was 1.5 ng/mL of plasma. Urine samples were directly injected and chromatographed on a microbore C18 column with an ion-pairing mobile phase. Excellent linearity was observed between the peak areas and concentrations in urine. The lowest limit of detection (signal:noise, 3:1) was 75 ng/mL of urine. The assay was used to determine the main pharmacokinetic parameters in healthy humans.