In traditional Chinese medicines, atractylodes rhizome ("Cangzhu" in Chinese) is used for the treatment of rheumatic diseases, digestive disorders, mild diarrhea, night-blindness. According to the Pharmacopoeia of China (2005 Edition), Rhizoma Atractylodis is the dried root of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. Polyacetylenes is one group of the most important active components in Cangzhu. A reversed-phase high performance liquid chromatographic method was developed for the simultaneous determination of two main polyacetylene components including atractylodin and atractylodinol in Cangzhu. The chromatographic analysis was carried out using a Polaris-C18 column and the mobile phase of acetonitrile-water with the flow rate of 1.0 mL/min. The detection wavelength was set at 340 nm, and column temperature was set at 25 degrees C. The detection limits (S/N = 3) were 0.069 and 0.016 microg for atractylodin and atractylodinol, respectively. The recoveries of the two polyacetylene components were found in the range of 97.4% - 104.6%. Thirteen samples collected were determined which includes three species of atractylodes rhizome, A. lancea, A. chinensis, and A. japonica. This rapid and accurate method has been successfully applied to the simultaneous determination of the two polyacetylene components in Cangzhu.