Hamster eggs with follicular cells were fertilized by epididymal spermatozoa in two chemically defined media. The proportion of penetrated eggs was significantly higher in a medium for rabbit (16%) than in a medium for rat eggs (6%), but much lower than in Tyrode's solution containing follicular fluid or blood serum as reported by others. The optimal sperm concentration for sperm penetration ranged from 0·5 to 5 × 106/ml but penetration of denuded eggs failed in these media. When exposed to hamster spermatozoa in the rabbit medium containing living or dead spermatozoa of guinea-pig, rat, mouse or hamster, high proportions of denuded eggs (24-96 %) and eggs with follicular cells (93%) were penetrated. By exposure of denuded hamster eggs to hamster spermatozoa in supernatant fluid of frozen-thawed guinea-pig spermatozoa, 97% of eggs were penetrated in 8 hr compared to 0% in the control group. Sperm capacitation was also efficiently induced by preincubation of hamster spermatozoa in the supernatant fluid. The fertilizing capacity of hamster spermatozoa was maintained for 12 hr during incubation with frozen-thawed guinea-pig spermatozoa when the concentration of hamster spermatozoa ranged between 10 and 20 × 106/ml. The beneficial factor of guinea-pig spermatozoa appeared to be from spermatozoa themselves, not from the vasal or epididymal fluids. The presence of follicular cells, blood serum, bovine serum albumin, or even polyvinylpyrrolidone in the media is essential for the capacitation and acrosome reaction of hamster spermatozoa. The components of guinea-pig spermatozoa appear to maintain the fertilizing capacity of hamster spermatozoa and stimulate the process of capacitation.