Trout spermatozoa are very sensitive to freeze-thawing, and the best cryoprotectants tested until now have a highly variable protective effect. It is not rare that only 10% of the frozen spermatozoa display an undamaged plasma membrane. The aim of this study was to determine whether membrane fragility of rainbow trout sperm (Oncorhynchus mykiss) is due to membrane lipid phase transitions, as postulated for other species, and to explore stabilization of membrane phospholipids by the components present in the freezing extender. Using Fourier transform infrared spectroscopy, we showed that the plasma membrane exhibited a steady decrease in fluidity as temperature decreased. A clear phase transition was observed only with purified membrane phospholipids. Stability of frozen-thawed liposomes made with trout plasma membrane phospholipids was assessed. Although dimethyl sulfoxide stabilized the liposomes better than glycerol, it showed negative interactions with other components added to the extender such as TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) or phosphate. We propose that membrane phospholipid liposomes provide an interesting way to assess the compatibility between various molecules when testing a freezing extender.