In vitro percutaneous delivery of hepatitis B vaccines was investigated in order to assess the penetration of vaccine under passive diffusion and iontophoresis conditions.  The study was carried out using Franz vertical diffusion cell through the hairless abdominal skin of Sprague-Dawley (SD) rats.  Enzyme-linked immunosorbent assay (ELISA) was used to determine the cumulative amount of permeation and the retention amount of drug in skin.  Passive diffusion alone resulted in less skin permeation and retention of hepatitis B vaccines, only (2.1 ± 0.1) ng·cm⁻² and (2.3 ± 0.1) ng·cm⁻² after 24 h when the initial concentration of vaccine in the donor compartment was 23 μg·mL⁻¹ and 46 μg·mL⁻¹, respectively.  After removing the stratum corneum,  the permeation and retention amount of hepatitis B vaccines increased to (383.7 ± 86.2) ng·cm⁻² and (16.8 ± 4.6) ng·cm⁻², respectively, 171.6-folds and 2.1-folds more than that from its intact skin with the drug loaded at 46 μg·mL⁻¹.  Iontophoresis induced a significant increase of cumulative and retention amount of hepatitis B vaccines through the skin (P < 0.05).  Application of iontophoresis significantly enhanced the permeation of hepatitis B vaccines (P < 0.05) by 2.7-folds and 6.6-folds for the intact skin, and by 1.6-folds and 1.8-folds for  the tape-stripped skin with initial drug loading of 23 μg·mL⁻¹ and 46 μg·mL⁻¹, respectively.  Iontophoresis also significantly increased the amount of drug retained in the skin.  After applying iontophoresis for 6 h, the amount of skin retention was nearly the same as passive diffusion for 24 h both from intact skin (16.8 ± 4.6) ng·cm⁻² vs (13.3 ± 5.4) ng·cm⁻² (P > 0.05) and tape-stripped skin (36.7 ± 14.1) ng·cm⁻² vs (26.8 ± 11.2) ng·cm⁻² (P > 0.05).  Overall, these findings revealed that the transportation efficiency of bioactive substance like hepatitis B vaccines may be improved by iontophoresis, which can be potentially used in the field of transcutaneous immunization.