The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 X 200 cm) in 0.05 M tris-NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HM alpha, alpha, beta H, beta L, gamma) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 x 10(6), 5.83 x 10(5), 1.58 x 10(5) , 4.59 x 10(4), 2.14 x 10(4) as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight alpha (HM alpha) and alpha, three for beta H, two for beta L and one for gamma. The gross CD patterns of crystallins were about the same in the peptide region (200 nm similar to or approximately 250 nm) with a minimum centered at about 217 nm, indicative of a beta-sheet structure in all crystallins. The [theta] values at 217 nm ranged from --1700 to --3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm similar to or approximately 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.