The single toxicity of diethylstilbestrol (DES) and beta-estradiol 17-valerate (EV) and the joint toxicity of their binary mixtures in equiconcentration to the proliferation of MCF-7 cells were investigated, respectively. Additive index (AI) method was adopted to evaluate the joint toxicity effect. At the same time, 3 x 3 factorial experimental design was used to verify the joint toxiciy types derived from equiconcentration of DES and EV. The results show that the EC50 values of single EV and DES for 24, 48 and 72 h are 6.02, 0.40 and 0.33 nmol x L(-1) and 5.90, 6.98 and 2.90 nmol x L(-1), respectively. The EC50 values of the binary mixtures of DES and EV for 24, 48 and 72 h are 2.33, 0.71 and 0.39 nmol x L(-1). The binary joint effects of DES and EV for 24 h were synergistic, and the joint effects of DES and EV for 48 and 72 h were antagonistic. But synergistic and antagonistic effects are not strong; their values can be found close to the values of additive effects. Factorial experiment results show that combined effects of DES and EV to proliferation of MCF-7 cells for 24, 48 and 72 h three exposure periods are additive effect types. The consistent joint combined effect types can be drawn from both factorial experimental design and equiconcentration ratio of DES and EV to the proliferation of MCF-7 cells. However, the factorial experimental design is simpler and more convenient, and can avoid unnecessary mistakes due to the derivation of EC50 values.