The mechanism of penetration into erythrocytes and metabolism of O, S-dicarbethoxythiamine (DCET) were investigated in vitro, using rabbit erythrocyte suspension. DCET readily entered into erythrocytes untill the concentration in the cell became equal with that in the medium, and this penetration was not affected by metabolic inhibitors such as monoiodoacetic acid or 2,4-dinitrophenol. After penetration into the cell, DCET was gradually converted to thiazole type metabolites in which thiamine and O-carbethoxythiamine were included, and accumulated in the cells because of low permeability of these metabolites. This metabolic reaction was suppressed when the erythrocytes were denaturated by heat treatment, or when the incubation was carried out at a low temperature. In addition, the relationship between the sub-strate concentration and the rate of metabolism was shown to satisfy the equation of Michaelis and Menten. These results suggested that DCET penetrates the erythrocyte membrane by a simple diffusion mechanism and is metabolized to thiamine by an enzyme present in the erythrocytes.