Our recent study has shown that betaA3-crystallin along with betaB1- and betaB2-crystallins were part of high molecular weight complex obtained from young, old, and cataractous lenses suggesting potential interactions between alpha- and beta-crystallins (Srivastava, O. P., Srivastava, K., and Chaves, J. M. (2008) Mol. Vis. 14, 1872-1885). To investigate this further, this study was carried out to determine the interaction sites of betaA3-crystallin with alphaA- and alphaB-crystallins. The study employed a mammalian two-hybrid method, an in vivo assay to determine the regions of betaA3-crystallin that interact with alphaA- and alphaB-crystallins. Five regional truncated mutants of betaA3-crystallin were generated using specific primers with deletions of N-terminal extension (NT) (named betaA3-NT), N-terminal extension plus motif I (named betaA3-NT + I), N-terminal extension plus motifs I and II (named betaA3-NT + I + II), motif III plus IV (named betaA3-III + IV), and motif IV (named betaA3-IV). The mammalian two-hybrid studies were complemented with fluorescence resonance energy transfer acceptor photobleaching studies using the above described mutant proteins, fused with DsRed (Red) and AcGFP fluorescent proteins. The results showed that the motifs III and IV of betaA3-crystallin were interactive with alphaA-crystallin, and motifs II and III of betaA3-crystallin primarily interacted with alphaB-crystallin.