The subunits of glutenin, the disulfide-bonded, high-molecular weight protein fraction of wheat, have been separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on large-pore silica-based columns. Glutenin was first isolated from bulk samples, and effects of dissociating, reducing and alkylating agents on subsequent RP-HPLC separations of glutenin subunits were compared. Eight RP-HPLC columns (Crs, Cs, diphenyl or cyanopropyl) were compared to achieve optimal resolution of glutenin subunits. Proteins were eluted by a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid and were detected at 210 nm. Excellent separations occurred when glutenin was dissociated in the presence of 8 M urea or 6 M guanidine hydrochloride, reduced with 5% 2-mercaptoethanol or 0.1% dithiothreitol, alkylated with 4-vinylpyridine and chromatographed on Crs or Ca bonded-phase columns. Using these conditions, fifteen to twenty major glutenin subunits were resolved. Glutenin subunits were then extracted from single kernels of aneuploid lines of the variety Chinese Spring and fractionated by RP-HPLC. Four early eluting peaks were shown to contain major glutenin subunits coded by the long arms of chromosomes 1D and lB, and thus were assumed to correspond to the high-molecular-weight subunits. Their elution volume indicated that these subunits have lower surface hydrophobicities than do most lower-molecular-weight glutenin subunits which eluted later. Significant differences in glutenin subunit composition among bread wheat varieties suggest possible relationships between specific polypeptides and breadmaking quality, and show that RP-HPLC of glutenin subunits has the potential of identifying most wheat varieties. These results demonstrate that RP-HPLC is a valuable complement to other chromatographic and electrophoretic methods for analysis of glutenin subunits.