The close association of hepatitis B antigen with serum proteins was demonstrated. The small morphological form of the antigen, measuring approximately 20 nm in diameter, was separated by isoelectric focusing and the heterogeneity of the antigen was confirmed by the finding of unique polypeptides in each preparation. Comparative trace-labelling techniques showed the integral nature of the constituent polypeptides, two of which were found to be mutually exclusive in the separated particles. Release of these polypeptides was achieved by treatment with Nonidet P40 and 2-mercaptoethanol. The significance of these findings in relation to hepatitis B virus is discussed together with the importance of surface antigenic variation as a function of protein heterogeneity of this virus.