The distribution of 3-β-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-β-hydroxysteroid-specific probe filipin. This non-destructive technique allows a spatially very detailed study of the distribution of 3-β-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-β-OH-cholesterol distribution along the optical axis. The 3-β-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-β-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-β-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.