Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm-1 divided by the Raman intensity at 2935 cm-1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens.