Despite the use of bovine gamma-crystallins in numerous biophysical and chemical studies, characterization of these proteins at the molecular level is incomplete. Bovine lenses have at least six gamma-crystallin protein fractions currently assigned as gamma s/I, gamma A/IVb, gamma B/II, gamma C/IIIb, gamma D/IIIa and gamma E/IVa. A lack of primary sequence data for corresponding gamma-crystallin genes and proteins, however, has made these assignments tenuous. To clarify these assignments, we have over-expressed recombinant bovine gamma-crystallin proteins in Escherichia coli using complementary DNAs corresponding to gamma B-, gamma C-, and gamma D-crystallin genes. The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions gamma II, gamma IIIa and gamma IIIb. The elution of recombinant gamma B and native gamma II proteins was identical on cation-exchange chromatography as expected; however, recombinant gamma C coeluted with gamma IIIa and recombinant gamma D co-eluted with gamma IIIb. Sequential Edman degradation through the first 29 residues of the native gamma IIIa and gamma IIIb polypeptides confirmed the colinearity of their sequences with those predicted from the gamma C- and gamma D-crystallin genes, respectively. Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for gamma-crystallins. Based on these data the bovine gamma-crystallins proteins and genes are correlated as follows: II/gamma B, IIIa/gamma C and IIIb/gamma D. The assignment of gamma IIIb (previously characterized as having a low critical temperature for phase separation) with gamma D rather than gamma C proves an exception to the hypothesis that the gamma ABC-crystallin group is more resistant to phase separation than the gamma DEF group. These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics. Additionally, expression of recombinant gamma-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the gamma-crystallins.