Isocratic high-performance liquid chromatography on PLRP-S 8-microns poly(styrene-divinylbenzene) copolymer allows complete separation of oxytetracycline, 4-epioxytetracycline, tetracycline, anhydrooxytetracycline, alpha- and beta-apooxytetracycline. The mobile phase was tert.-butanol-0.2 M phosphate buffer pH 8.0-0.02 M tetrabutylammonium sulphate pH 8-0.0001 M sodium ethylenediaminetetraacetate pH 8.0-water (5.9:10:5:10:78.1, m/v/v/v/v). With this isocratic method, 2-acetyl-2-decarboxamidooxytetracycline is only partly resolved from oxytetracycline. The separation and the detection limits can be improved by the use of gradient elution. Gradient elution was used for the comparison of official standards and for the analysis of a number of commercial samples, and to monitor the stability of oxytetracycline hydrochloride during storage in the solid state for about 6 years at various temperatures.