The lipid composition of hepatitis B surface antigen (HBsAg) (subtype adw) obtained from different carriers has been determined and proven to be truly characteristic of HBsAg and not subject to individual variation. Phosphatidylcholine (approximately 60%), cholesteryl ester (approximately 14%), cholesterol (approximately 15%), and triglycerides (approximately 3%) are the main HBsAg lipid constituents. The fatty acid composition of the different HBsAg lipid components is similar to that of other normal human serum lipoprotein. A photoactivatable hydrophobic probe, pyrenesulfonyl azide, has been used to determine what portions of the protein components of HBsAg are exposed to the HBsAg lipid matrix. Both major HBsAg protein components became randomly pyrenesulfonyl azide labeled in both the NH2-terminal and COOH-terminal tryptic fragments, therefore suggesting they are buried within the HBsAg lipids. A model for the arrangement of proteins in HBsAg is proposed whereby regions within the NH2-terminal and COOH-terminal parts of the two major HBsAg protein components are buried within the lipid matrix of HBsAg particles, while the antigenically important residue 122-150 region is exposed to the aqueous environment.