Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. Betagamma-crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-crystallin for calcium-binding studies. Using calcium-binding assays such as 45Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both betaB2- and betaA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N-1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.