The a-crystallins are evolutionarily related members of the small heat shock protein (sHSP) superfamily which are taxonomically ubiquitous components of the vertebrate eye lens [1]. The aA-crystallin and aB-crystallin genes arose through a gene duplication event that occurred early in vertebrate history and are most closely related to sHsp20 [2]. Mammalian aA-crystallin is primarily lens specific and has lost the stress induction response that characterizes most sHsps, although some metals induce its expression [3]. In contrast, multiple cellular stresses induce mammalian aB-crystallin expression in a variety of tissues [4]. The mammalian a-crystallins act as chaperone-like molecules by binding to and preventing the aggregation of non-native We previously reported that zebrafish aB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish aBcrystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel a-crystallin transcript designated cryab2 and the resulting protein aB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human aB-crystallin and zebrafish aB1-crystallin, respectively. RT-PCR showed that aB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian aB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant aB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of a-lactalbumin and lysozyme. At 25°C and 30°C, zebrafish aB2 showed greater chaperone-like activity than human aB-crystallin, and at 35°C and 40°C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that aB2-crystallin makes up 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different aB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish aB-crystallins perform divergent physiological roles. After gene duplication, zebrafish aB2 maintained the widespread protective role also found in mammalian aB-crystallin, while zebrafish aB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.Abbreviations sHSP, small heat shock protein.