Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca²⁺), phosphate (PO₄^3-) and lactate. In all media containing no Ca²⁺, including medium lacking Ca²⁺, lacking Ca²⁺ and PO₄^3-, lacking Ca²⁺ and lactate and lacking Ca²⁺, PO₄^3- and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca²⁺ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca²⁺. In conclusion, preincubation of thawed sperm in medium containing no Ca²⁺ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca²⁺ is practical for use in cryopreserved C57BL/6J sperm.