A sensitive and reproducible assay employing liquid-liquid extraction and high-performance liquid chromatography with fluorescence detection for the quantification of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and Z-4-hydroxy-N-desmethyltamoxifen in human plasma is described. The compounds and internal standard, propranolol, were separated with a cyano column and a mobile phase of acetonitrile-20 mM potassium phosphate buffer (pH 3; 35:65, v/v) then detected with fluorescence using a modified version of a method originally described by Fried and Wainer [J. Chromatogr. B 655 (1994) 261]. The coefficients of variation for the midpoint of the standard curve for each compound were less than 10%. This method was applied to a pharmacokinetic study of tamoxifen disposition in breast cancer patients.