Macropod spermatozoa have proven difficult to cryopreserve such that empirical studies using high concentrations of glycerol and/or DSMO have resulted in only 10% post-thaw motility. We examined the ultrastructure and freeze-fracture of caput and cauda epididymal macropod spermatozoa at 35, 4 degrees C and following cryopreservation with and without 20% glycerol. The addition of 20% glycerol resulted in significant damage to the sperm plasma membrane and mitochondria compared to no glycerol at the same temperatures (P<0.05). Following cryopreservation, 20% glycerol significantly improved the preservation of the cauda epididymal sperm plasma membrane and mitochondria and reduced the incidence of axonemal damage and axonemal spaces. For caput epididymal spermatozoa, glycerol only improved the preservation of the plasma membrane following cryopreservation (P<0.05). Freeze fracture microscopy revealed a pattern of helically wound intramembranous particles in the plasma membrane over the fibre network of the mid piece of the sperm tail. The fibre network is an interconnecting cytoskeletal structure found underneath the plasma membrane of the kangaroo sperm midpiece and is thought to add rigidity to the proximal portion of the sperm tail. After thawing, the plasma membrane was damaged such that this structure was missing in patches, and the helical rows of particles were mal-aligned. On the principal piece, particles were arranged randomly at physiological temperatures; however, upon cooling to 4 degrees C with 20% glycerol, the particles become aggregated. Once rewarmed (35 degrees C), particles over the principal piece resumed their random organisation. This finding is further evidence of a reversible phase transition of the macropod sperm plasma membrane during cooling that is not associated with a loss of motility or membrane integrity.