In vitro fertilization of mouse, golden hamster, and rat eggs in media at various pH values was investigated. A modified Krebs—Ringer bicarbonate solution, a modified Tyrode’s solution with heated hamster blood serum, and a modified Krebs—Ringer bicarbonate solution with heated rat blood serum were used, respectively, for the mouse, hamster, and rat gametes. Although sperm penetration could occur in media between pH 6 and 8.5, 6.7 and 8.7, and 6.75 and 8.3, the optimal pH ranged from 7.3 to 7.7, 6.8 to 8.2, and 7.8, respectively, for the penetration of mouse, hamster, and rat eggs. It seems that in vitro fertilization of rat eggs can be achieved in media only within a relatively narrow range of pH higher than pH 7, while hamster and mouse gametes can withstand a relatively wide range of pH.