During incubation of guinea-pig spermatozoa in Ca-free Krebs—Ringer phosphate buffer, substances were released into the medium which were subsequently separated on Sephadex G-200 into two major fractions, Fr. I and Fr. II. The macromolecular Fr. I increased with sperm maturation and the fraction was shown to possess four different antigens. The amount of Fr. I. released from spermatozoa during incubation in the uterus was apparently influenced by the hormonal environment of the female reproductive tract. The fraction showed decapacitating activity, and decapacitated spermatozoa treated with Fr. I were capable of recapacitation after a second uterine incubation period. It was concluded that Fr. I was the decapacitation factor in guinea-pig semen.