Didelphis spermatozoa released from different levels of the epididymis exhibit a series of changes that reflect events involved in the process of their maturation. Their progression through the duct is accompanied by a rotation of the sperm head on the axis of the tail, by an eccentric movement of the cytoplasmic droplet to the anterior region of the midpiece from where it is discarded, by appearance in the posterior midpiece of regular submembranous electron-dense ridges, and by visible condensation of the mitochondrial cristae and matrix. The latter seems to correlate generally with onset of the ability for progressive motility acquired by most spermatozoa as they reach the lower corpus epididymidis. This phase is associated also with the beginnings of the pairing by the acrosomal surface that characterizes mature spermatozoa of New World marsupials. Use of lectins and cationized ferric colloid as affinity markers served to reveal changes occurring also in the character of the sperm head and tail surface prior to pairing. This investigation strengthens the idea gained from previous study of an Australian marsupial, Trichosurus, that the complexity of sperm maturation in the epididymis of Metatheria is broadly comparable to that observed in most eutherian mammals.