The soluble lens proteins (a. p, and y crystallins) of the adult American grass frog, R. pipiens, were investigated, using DEAE-cellulose ion-exchange chromatography and disc electrophoresis on polyacrylamide gels. Column chromatography of whole adult lens proteins, with a NaHzP04-NazHP04-NaCl buffer elution program, revealed six fractions. The first to be eluted was identified as y crystallin, the predominant lens protein of this organism; the second and third as , 8 crystallins; and the fourth, fifth, and sixth as a crystallins. Disc electrophoresis of these fractions on polyacrylamide gels of appropriate porosity confirmed the identity of these proteins as separated by ion-exchange chromatography. Rechromatography of the isolated y crystallin fraction of whole lens protein, employing a tris-HC1 buffer elution system, resolved the y crystallin into four distinct components. These individual y crystallins exhibited different electrophoretic mobilities, as judged by their behavior in disc electrophoresis. This precludes the possibility that the fractions obtained are artifacts of the elution process, and confirms the existence of four y crystallins in the lens of adult R. pipiens, as has been reported for mammalian (bovine) embryonic and adult y crystallins. The phylogenetic and developmental implications of these findings are discussed.