The purpose of this study was to determine whether a 9 kDa gamma D-crystallin fragment, on in vivo post-translational modifications, exists as isoforms in the water soluble protein fraction of human lenses. In this study, three isoforms of the 9 kDa polypeptide (named as 9 kDa I, II and III) were identified and purified. In addition, the possible modified amino acids and their locations in the three isoforms were identified. The purification of the three isoforms was achieved by four steps which included separation of a mixture of crystallin fragments from the intact crystallins by a Sephadex G-50 chromatography under denaturing conditions, followed by purification of the 9 kDa polypeptide isoforms by a non-denaturing gel electrophoresis, preparative SDS-PAGE and HPLC using a C-18 column. Each of the isoforms showed a single protein band and a single peak during SDS-PAGE and HPLC analyses respectively. The three isoforms on their partial N-terminal sequence analyses, exhibited sequence identical to gamma D-crystallin starting at residue no. 87 suggesting that the isoforms contained residues no. 87 to 173 of gamma D-crystallin. On comparison of the amino acid compositions of the isoforms with that of the identical 9 kDa gamma D-crystallin fragment, the isoforms showed relatively lower amino contents of Asp, Arg, Leu and Tyr residues suggesting modifications of these residues in the isoforms. To identify the specific regions at which these amino acid residues were modified, the Western blot analysis with six site-specific polyclonal antibodies to six regions of the 9 kDa gamma D-crystallin polypeptide was carried out. Of the six antibodies raised, one was to the N-terminal region (residue nos 87-95; named as anti-9 kDa N-Ab), second to the C-terminal region (residue nos 165-173; named as anti-9 kDa C-Ab) and four to the four different middle regions [named as anti-9 kDa M1 (nos 94-100)-Ab, M2 (nos 114-120)-Ab, M3 (nos 137-143)-Ab and M4 (nos 149-154)-Ab] of the polypeptide. The Western blot analysis suggested that the 9 kDa I and 9 kDa II isoforms had modified amino acid residues in the regions of residue nos 114-120 and 165-173 whereas the 9 kDa III isoform in the regions of residue nos 114-120, 137-143, 149-154 and 165-173. It was also determined whether the 9 kDa isoforms exhibit an age-related appearance in human lenses. Western blot analysis as above of the WS-proteins from lenses from donors of different ages was carried out. On comparison of these results with an identical Western blot analysis of the three purified 9 kDa isoforms, I, II and III, it was inferred that the 9 kDa isoform III appeared earlier than other isoforms during aging in human lenses.