Purpose. Phospholipids are a major component of lens fiber cells and influence the activity of membrane proteins. Previous investigations of fatty acid uptake by the lens are limited. The purpose of the present study was thus to determine whether exogenous fatty acids could be taken up by the rat lens and incorporated into molecular phospholipids. Methods. Lenses were incubated with fluorescently labeled palmitic acid and then analyzed by confocal microscopy. Concurrently, lenses incubated with either fluorescently labeled palmitic acid or the more physiologically relevant 13C18-oleic acid were sectioned into nuclear and cortical regions and analyzed by highly sensitive and structurally selective electro-spray ionization tandem mass spectrometry techniques. Results. The detection of fluorescently labeled palmitic acid, even after 16 hours of incubation, was limited to approximately the outer 25% to 30% of the rat lens. Mass spectrometry also revealed the presence of free 13C18-oleic acid in the cortex but not the nucleus. No evidence could be found for incorporation of fluorescently labeled palmitic acid into phospholip-ids; however, a low level of 13C18-oleic acid incorporation into phosphatidylethanolamine (PE), specifically PE (PE 16:0/13C18 18:1) was detected in the lens cortex after 16 hours. Conclusions. These data demonstrate that uptake of exogenous (e.g., dietary fatty acids) by the lens and their incorporation into phospholipids is minimal, most likely occurring only during de novo synthesis in the outermost region of the lens. This finding adds support to the hypothesis that once synthesized there is no active remodeling or turnover of fiber cell phospholipids. © 2011 The Association for Research in Vision and Ophthalmology, Inc.