The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4°C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classes and only small changes were observed in phospholipid classes of seminal plasma. The motility, viability, and morphological integrity of spermatozoa decreased during storage. These changes in phospholipid content may be explained by membrane phospholipid lysis followed by endogenous metabolism or by a complex combination of lysis, metabolism, and peroxidation. They are likely to affect semen quality and the success of in vitro storage severely.