PURPOSE. The purpose of this study is to compare the protein composition of the B-3 line of transformed human lens epithelial (HLE) cells to that of freshly dissected HLE cells, This provides baseline data on lens cell proteins from fresh lens cells and from the B-3 cell line, which is often used as a model system for the lens. METHODS. Human lens epithelial cells adherent to the lens capsule were dissected into central (undifferentiated) and peripheral (partially differentiated) populations. Fully differentiated human lens fiber cells were isolated from the outer cortical layers of the lens. HLE B-3 cells were analyzed at several passage levels. Extracts were prepared from each cell type and the proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). Representative gel patterns were visually compared, spots excised, and trypsin digests prepared. The peptide compositions of the digests were analyzed using either liquid chromatography electrospray ionization tandem mass spectrometry or atmospheric pressure-matrix-assisted laser desorption ionization mass spectrometry, using a liquid chromatography classic ion trap (LCQ) mass spectrometer. RESULTS. Two-DE patterns were obtained for fresh and cultured cell types. Similar patterns were observed between central and peripheral HLE cells, both of which contained high levels of αA-, αB-, and βB2-crystallins; α-enolase; and aldehyde dehydrogenase. HLE B-3 cultured cells were characterized by a marked loss of crystallins and a relatively higher level of noncrystallin proteins-most notably, high molecular weight, acidic proteins. Whereas subunit d of adenosine triphosphate (ATP) synthase, αB-crystallin, galectin, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, actin, peptidylprolyl isomerase A, phosphatidylethanolamine-binding protein, and vimentin were present in both fresh and cultured lens epithelium, only the high abundance of α-enolase, galectin-1, and vimentin suggested that B-3 cells were lens derived. CONCLUSIONS. Freshly dissected noncultured HLE cells from both central and peripheral regions contain a high concentration of crystallins that mask the detection of less abundant proteins by 2-DE, Transformation and culture of HLE cells causes a loss of these crystallins and an increase in the relative concentration of other proteins. However, most of these noncrystallin proteins were different from those observed in non-cultured HLE cells. These results suggest that transformation markedly alters the protein expression pattern in immortalized HLE cells and that caution should be exercised when using them to study properties of HLE cells in vivo.