Recently ovulated eggs of mice, rats, and hamsters were treated with hyaluronidase and subsequently with trypsin or pronase to disperse the corona radiata and zona pellucida. When these zona-free eggs were inseminated with epididymal or uterine sperm of rats, epididymal sperm of hamsters or mice in the presence of either heated bovine follicular fluid or heated blood serum and incubated for 1–12 hr, low proportions of mouse eggs were penetrated by rat or hamster sperm (1.3–5%), rat eggs by hamster sperm (9%) and hamster eggs by rat sperm (8.7–26%). Although enlargement of the sperm head was observed in these cases, the transformation of the sperm head into a male pronucleus did not occur, the activation of eggs to resume the second maturation division was rare, and the formation of the female pronucleus never occurred. Very high proportions of rat eggs (95%) or hamster eggs (73%) were penetrated by mouse sperm and polyspermy occurred as incubation time increased. In these cases, the transformation of many sperm heads into male pronuclei, the initiation of the second maturation division, and the formation of a female pronucleus occurred normally. The transformation of mouse sperm heads into male pronuclei in the rat eggs appeared to be earlier than that of mouse sperm in the hamster eggs. Although the incorporation or fusion of eggs with foreign sperm may depend on the possibility of sperm capacitation, physiological affinity between the sperm of one species and the vitellus of another is also an important controlling factor, but the zona pellucida appears to be the major block for interspecific fertilization.