This study was conducted to evaluate the pretreatment effects of different in vivo moxibustion on the permeation of a model high molecular compound, FITC-dextran, with a mean molecular weight of 4 kDa (FD-4), through excised hairless rat skin. Direct or indirect moxibustion (0.10 g moxa) was pretreated consecutively 4 times every 5 min on the abdomen of hairless rats, and the permeation of FD-4 was determined through the excised skin over 8h from 30 min after starting the first moxibustion. This consecutive moxibustion pretreatment showed a significant increase in the skin temperature as well as skin permeation of FD-4 compared with the control group (no moxibustion pretreatment). Quantitative parameters showed an increase in skin temperature and skin permeation: the area under the skin temperature over control temperature-time curve during one burning cycle (5.0 min) (AUCtemp) or the maximum skin temperature during moxibustion (Tmax) and the cumulative amount of FD-4 permeated through skin over 8h (Q8) or steady-state flux were increased by moxibustion pretreatment. Then, the effect of pedestal thickness (distance from the moxa cylinder and skin surface), shape of the moxa cylinder (5mm diameter, 13 mm height or 9 mm diameter, 7 mm height), burning materials (moxa or aromatic incense), pedestal component (paper, potato or ginger) and moxibustion pretreatment method (direct or indirect moxibustion) was evaluated on the AUCtemp or Tmax and Q8 or flux. The amount of protein leached from the skin surface was also determined as an inflammatory index by this moxibustion pretreatment. When the skin temperature was increased to 60 degrees C, the Q8 or flux as well as the amount of protein leached were markedly increased. When the skin temperature was controlled to 42 to 45 degrees C by an adequate selection of pedestal thickness, shape of the moxa cylinder, burning materials, pedestal component and moxibustion pretreatment method, on the other hand, protein leaching remained unaltered, but the Q8 or flux significantly increased with the Tmax. This study thus provides credible evidence that moxibustion pretreatment increases the skin permeation of high molecular compounds.