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The objective of this work is to investigate the percutaneous permeability of 2,3,5,6-tetramethylpyrazine (TMP), an active ingredient originally isolated from Ligusticum wallichii Franch. Certain physicochemical properties of TMP, including its partition coefficient and pH–solubility profile, were studied. The influence of pH on the percutaneous permeation of TMP was studied in vitro using hairless mouse skin. Comparative in vitro permeability of TMP through hairless mouse, rat, rabbit, and human cadaver skin was also investigated. The results indicate that hairless mouse skin and rat skin were about three to four times more permeable to TMP than human cadaver skin. The permeability of TMP through rabbit skin was not significantly different from that of human cadaver skin. The observed lag times for all skin membranes were about 1–2 h. Although pharmacokinetic data are not currently available to permit precise calculation of a clinically effective patch size, the data from this study indicate that the transdermal delivery of TMP should nevertheless be possible.