In an effort to investigate the nature of the cellular injury caused when mammalian spermatozoa are cooled prior to cryopreservation, the occurrence of thermal phase transitions amongs the lipid components of the sperm plasma membrane was investigated by the use of freeze-fracture electron microscopy. The mechanisms by which glycerol and egg yolk exert protective effects during semen cooling and freezing were also examined. Ram and blackbuck spermatozoa, maintained at 30 degrees C prior to fixation at this temperature, exhibited randomly distributed intramembranous particles over the acrosomal, postacrosomal, and flagellar regions of the plasma membrane. In contrast, spermatozoa fixed at 5 degrees C after slow cooling to this temperature exhibited particle clustering over the postacrosomal region of the head as well as over the tail. These effects were not influenced by the presence of egg yolk or glycerol during the cooling procedure, although these substances protected the spermatozoa against loss of motility. Particle clustering over the sperm tail, induced by the slow cooling process, was found to be only partially reversible. The extensive areas of particle-free lipid, noted to result from the cooling procedure, were absent if the spermatozoa were rewarmed to 30 degrees C; however, the original distribution of particles was not restored and numerous small particle-free domains persisted. It is proposed that this type of irreversible change within the sperm plasma membrane may contribute to the loss of motility and fertility suffered by spermatozoa after cooling and freezing. Furthermore, it is suggested that protective substances such as egg yolk may exert their effects by countering these deleterious changes, rather than by preventing their occurrence.