Abstract: ICR mouse blastocysts and morulas were cultured in rat heart conditioned medium (RH-CM).As far as the following indexes attaching ratio，ICM growth ratio and ES clone formation ratio are concerned，the choice of blastocysts is much better than morulas (P＜0.05)，therefore mouse blastocysts were more suitable than morulas to get ES cells. Interval between two passages (38 h) of expriment groups was significant higher than control group (P＜0.05). It was showed that RH-CM not only could maintain ES cells in the undifferentiated state, inhibiting the spontaneous differentiation effectively, but also promote adherence and proliferation of mouse ES cells. The effect of the three culture systems on formation of ES colonies at primary passage was not remarkable but significant in ES colonies formation after passage and the highest times of passage. The combination of RH-CM with mitotically inactive MEF feeder layer was the best culture system.