Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification andquantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel directinsertionelectron-ionization technique found the cholesterol content of human lenses to be significantlyhigher (ca. 6 times) than lenses from the other animals.The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat,mouse, sheep, cow, pig and chicken, thesewere present largely as phosphatidylcholines, in contrast 66% of thetotal phospholipid in Homo sapienswas sphingomyelin, with the most abundant being dihydrosphingomyelins,in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenseswere found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly highconcentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify thephospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantifyindividual lens phospholipids using internal standards. These data clearly indicate marked differences in themembrane lipid composition of the human lens compared to commonly used animal models and thus predict asignificant variation in the membrane properties of human lens fibre cells compared to those of other animals.