Purpose: The purpose was to characterize the properties of a proteinase activity associated with βA3-crystallin, which was isolated from the α-crystallin fraction of human lenses. Methods: An inactive, Arg-bond hydrolyzing proteinase in the α-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant γB-, γC-, and γD-crystallins, and its presence in three different protein fractions of human lenses (i.e., α-crystallin, βH-crystallin, and membrane fractions). Results: An inactive, Arg-bond hydrolyzing proteinase present in the α-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl β-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the α-crystallin fraction since it eluted at a lower molecular weight species than α-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as βA3-, βB1-, and βB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant βA3-, βB1-, or βB2-crystallins, only the βA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of γC- and γD- crystallins, and the cleavage of γD-crystallin at M1-G2, Q54-Y55, M70-G71, and Q103-M104 bonds. Further, the enzyme was also present in three fractions of human lenses (α-crystallin, βH-crystallin, and membrane fractions). Conclusions: A serine-type βA3-crystallin proteinase existed in an inactive state in the α-crystallin fraction and was activated by detergents. The enzyme proteolyzed αA-, αB-, γC-, and γD-crystallins and was present in three fractions (α-crystallin, βH-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses. © 2008 Molecular Vision.