Summary. Oocytes, recovered from the oviduct or from ripe follicles, and spermatozoa flushed from the oviduct of opossums mated under laboratory conditions, were used to study the characteristics of marsupial gamete interaction and sperm incorporation. Opossum oocytes lose all granulosa cells before ovulation and are invested only by a thin zona pellucida when ovulated. The spermatozoa unpair in the oviduct but the motile population flushed at about the time of ovulation includes paired, separating and single spermatozoa. Separation of paired spermatozoa was associated with changes in the acrosome, the amorphous matrix being displaced by membrane-bound vacuoles. Fertilization is normally monospermic. Spermatozoa associated with the zona of unfertilized eggs by the flat acrosomal face of the head, whereas those trapped in mucoid eventually laid down around the zona were orientated at random. In penetrating the zona, the spermatozoon created a relatively large uneven hole that contrasts with the discrete penetration slit seen in the thicker zona of eutherian oocytes. Spermatozoa appeared to associate and fuse with the oolemma by the acrosomal face of the head. Ultrastructurally the sperm head, in oocytes fixed immediately after incorporation into the ooplasm, was devoid of surrounding membranes. This pattern of gamete interaction resembles the mode seen in non-mammalian vertebrates and invertebrates, and not that in eutherian mammals. Sperm penetration by tubal sperm samples occurred readily in vitro within 1 h of placing ripe follicular oocytes with the oviducal flushings containing spermatozoa. None of the oocytes penetrated in vivo or in vitro displayed extra perivitelline spermatozoa. This suggests that the oocyte is able to mount a block to polyspermy at the zona surface, although the mucoid deposited on the zona pellucida may be involved in vivo . The results indicate that the complex mode of sperm incorporation seen in eutherian mammals is unique to the infraclass and not shared by their closest relatives the marsupials.