The relationships of the various polypeptides associated with hepatitis B surface antigen (HBsAg), ground squirrel hepatitis surface antigen (GSHsAg), woodchuck hepatitis surface antigen (WHsAg), and duck hepatitis B surface antigen (DHBsAg) were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tryptic peptide mapping. Analysis of independent antigen isolates by SDS-PAGE resulted in bands consistently observed at 24,000, 28,000, 32,000, 43,000, and 50,000 Da with HBsAg; at 22,000, 25,000, 35,000, 37,000, 39,000, and 42,000 Da with GSHsAg and WHsAg; and at 18,500, 30,000, and 38,500 Da with DHBsAg. Comparison of the major polypeptide pair from the mammalian viruses by tryptic peptide mapping suggests more than a single point of glycosylation or other post-translational modification(s) in some paired comparisons and/ or heterogeneity in glycosylation in others. Comparison of the major component of each mammalian virus (HBsAg p24, GSHsAg p22, or WHsAg p22), or the major polypeptide of DHBsAg (p18.5), with their respective larger polypeptides by peptide mapping indicated that one or more of the larger components in each virus shares extensive homology with the appropriate major component. Further, these larger components possess additional spots, interpreted as additional primary sequences, which were not found in the map of the appropriate major component. Collectively, the results suggest that a number of surface antigen-associated polypeptides may be partially encoded for by the pre-S gene region known to exist in hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV), and likely to exist in ground squirrel hepatitis virus (GSHV) and duck hepatitis B virus (DHBV) DNA.