I reported previously that cell malignancy was promoted significantly by treatment with 3'-Me-DAB in the presence and absence of S15 mixture. In this report, I examined the effect of treatment frequency of 3'-Me-DAB in the presence and absence of S15 mixture on a cloned rat liver cell line (CL-20) which had low-tumor producing capacity. Tumors produced by subcutaneous back-transplantation of CL-20 cells treated with 3'-Me-DAB in the presence and absence of S15 mixture showed more undifferentiated, solid and infiltrative morphology than that of non-treated control cells. The tumors produced by back-transplantation of CL-20 cells treated with high concentrations of 3'-Me-DAB showed metastatic growth in the lung of rats, but those produced by back-transplantation of untreated CL-20 cells did not. Plating efficiency increased in proportion to the treatment frequency of 3'-Me-DAB in the presence and absence of S15 mixture. This fact suggests that plating efficiency is effective as a marker for the promotion of 3'-Me-DAB-induced cell malignancy. The aggregate sizes formed by the rotation culture were larger in the cells treated with 3'-Me-DAB in the presence and absence of S15 mixture than the control cells. However, the effect of the treatment frequency of 3'-Me-DAB was rarely found in this experiment. Few changes were observed with other in vitro markers for the promotion of cell malignancy. These findings suggested that cell malignancy could be promoted by repeated treatments of 3'-Me-DAB in the presence or absence of S15 mixture in vitro.