Three classes of monomeric crystalline separable by SDS gel electrophoresis or by gel filtration have been demonstrated in human lens. In previous studies we have concluded from physico-chemical and immunological data that all three polypeptides are related and should be classified asγ-crystallins. The present paper presents further evidence supporting this conclusion, but also demonstrates that the 24,000 dalton (24kD) polypeptide corresponds to theβS-crystallin.βS-crystallin was purified by classical techniques from bovine lens and was shown to cross-react with a monoclonal antibody specific for the human 24kD polypeptide. This antibody exhibited no reactivity to other crystallin fractions from either bovine or human lenses. The identification of the 24kD polypeptide asβSwas further supported by analysis of the tertiary structures of the molecules by near UV-circular dichroism and by the finding of a blocked amino terminus on the 24kD polypeptide. Our finding that the humanβS(24kD polypeptide) should actually be classified as aγ-crystallin is fully consistent with recently reported sequence data on bovineβS-crystallin.