Possum spermatozoa undergo a distinctive process of maturation in the epididymis, as shown by change in the properties of the sperm surface, by modification of their morphology and by their increasing capacity for progressive motility. Modification of the sperm surface over the head and tail is demonstrated by the different affinities of sperm from successive regions of the epididymis for FITC‐conjugated wheat germ agglutinin and concanavalin A, and for cationic ferric oxide colloidal particles. Changes in sperm head morphology are caused by (1) a dramatic reshaping and consolidation of the acrosome in which excess plasma membrane overlying it is sloughed as a cluster of vesicles, (2) a reorientation of the nucleus almost parallel to the axis of the tail and (3) distal movement of the droplet from its initial envelopment of the nucleus to an eccentric position on the anterior segment of the midpiece. Spermatozoa released from the testis and caput epididymidis are essentially immotile or exhibit only lazy uncoordinated movements, whereas many from the corpus and most from the more distal regions of the epididymis display an energetic, progressive motility imparted by a rapid and stiff tail beat of narrow arc. This maturation of the capacity for motility is accompanied by an enhanced stability of the dense fibers and sheath, which become more resistant to the disruptive action of SDS and DTT, and by changes in the ultrastructure of the sperm tail. These include modification of the matrix of the mitochondria and also an unusual differentiation of the midpiece into two distinct segments. The anterior segment is defined by profuse peri‐mitochondrial stacks of membranes which develop as spermatozoa pass through the epididymis. These membranes, although prominent in mature spermatozoa fixed in situ, appear sparse and disorganised in spermatozoa fixed after 15 to 30 minutes of active motility in physiological medium, suggesting their possible utilisation in motile spermatozoa. The posterior segment is characterised by a thick peri‐mitochondrial cytoplasmic sleeve, by spirally arranged parallel fibrous bands immediately beneath the plasma membrane and, subsequently, as spermatozoa pass into the lower corpus epididymidis, by rows of flask‐shaped surface invaginations which develop between the spiral bands. Despite broad similarities in the features of sperm maturation in this marsupial and in eutherian mammals, there are distinct differences in the structural organisation of their spermatozoa, particularly in the sperm head. Until more is known of the details of fertilisation in marsupials the significance of these differences will remain unclear.