Abstract Simultaneous determination of oxytetracycline, 4-epioxytetracycline, α-apooxytetracycline, tetracycline and β-apooxytetracycline on C 18 columns has been accomplished using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS RP-C 18 column (250 mm × 4.6 mm) and on a Waters C 18 Symmetry column (150 mm × 3.9 mm), 5 μm particle size each. These columns were equilibrated with mobile phases consisted of methanol–acetonitrile–0.1 M phosphate buffer pH 8.0 (12.5:12.5:75, v/v/v) and (15:15:70, v/v/v), respectively. The flow rate was 1.0 ml/min and the total elution time was 15 and 5 min, respectively. Both methods were applied to oxytetracycline raw material, human and veterinary formulations, where the excipients did not interfere. External standard calibration curves were linear for 4-epioxytetracycline, oxytetracycline, α-apooxytetracycline, tetracycline and β-apooxytetracycline in the concentration range of 0.27–200 μM, 0.05–200 μM, 0.03–200 μM, 0.35–200 μM and 0.20–200 μM on column A and 0.08–200 μM, 0.15–200 μM, 0.09–200 μM, 0.25–200 μM and 0.47–200 μM on column B, respectively. Day-to-day relative standard deviation of the determination for every component was less than 3%. Concerning the first column, limits of detection and quantification of the above compounds were in the concentration ranges of 10–106 nM and 30–352 nM, respectively, whereas on the second column these ranges became 27–144 nM and 81–475 nM, respectively. Recovery of the separated compounds was 95–105%.