Abstract: OBJECTIVE To establish an HPLC method for quantitative determination of tacrolimus in vitro percutaneous solution, and investigate the ability of percutaneous penetration of 0.03% Tacrolimus ointment. METHODS Diamonsil C₁₈ column(250 mm×4.6 mm, 5 μm) was used as the fixed phase and a mixture of acetonitrile, purified water and phosphoric acid(700∶300∶1) as mobile phase. The flow rate was 1.0 mL·min^-1. The detection wavelength was at 215 nm. Improved Franz type diffusion cells were used in vitro permeation studies and excised mini pigs skins in vitro were used as transdermal barrier. The concentration of tacrolimus in the receptor solution was determined by HPLC to investigate its cumulative permeation quantities at different time respectively and compared with that of the commercial ointment. RESULTS The regression equation of Tacrolimus ointment was linear in the range of 2.088-20.88 μg·mL^-1(A=16 310.263C+3 629.783, r =0.999 97). The average recovery was 99.10%. A good linear relationship existed between the cumulative penetration quantities and the time. There was no significant difference in the permeation quantities between the self-made and commercial ointment. CONCLUSION The method is simple, rapid, and accurate. It is a good method to evaluate the characteristic of permeation for Tacrolimus ointment.