Fourteen known phorbol diester constituents of Croton tighum L. seed oil (croton oil) have been separated by a two-dimensional thin-layer chromatographic (2D-TLC) technique using different sorbent layers on the same plate. After a preliminary purification step using low-pressure liquid chromatography, reversed-phase overpressure liquid chromatography (RP-OPLC) has been shown to be potentially useful for the rapid preparative purification of these toxins. The identity of the major croton oil phorbol diesters was established by cochromatography with authentic compounds and/or by off-line chemical ionization mass spectrometry. The standard tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, was obtained free from impurities as a result of the application of both the 2D-TLC and liquid chromatography-RP-OPLC procedures.