A high-pressure liquid chromatographic method has been developed for the quantitative analysis of tetracyclines from biological material. Tetracyclines can be extracted from serum or organ homogenates by means of acetonitrile containing buffer. The extract is chromatographed on a reversed-phase column and the tetracyclines are monitored spectrophotometerically at 357 nm. Tetracyclines, pyrrolidinomethyl-tetracycline, 4-epitetracycline, and doxycycline are thus separated within 8 min. The lowest concentration for the intact molecule detectable are 0.2 micrograms/ml serum or blood and 0.4 micrograms/g organ. In a comparative study the new extraction procedure and the chromatographic analysis yielded the same results as a microbiological method.