Amino acid substitutions of hepatitis B surface antigen (HBsAg) may affect the antigenicity and immunogenicity of HBsAg, leading to immune escape and diagnostic failure. The amino acid positions 122 and 160 are known as determinants for HBsAg subtypes d/y and w/r, respectively. The substitution K1221 has been shown to strongly affect HBsAg antigenicity. In this study, we investigated the significance of naturally occurring amino acid substitutions K1221, T123N, A159G and K160N. Both T123N and K160N substitutions resulted in additional N-glycosylated forms of HBsAg, while the other mutations produced more glycosylated HBsAg compared with the wild type (wt). Detection of HBsAg by ELISA and immunofluorescence staining indicated that variant HBsAg (vtHBsAg) with K1221 was not recognized by HBsAg immunoassays, while vtHBsAg with T123N, All 59G, K160N and A159G/K1SON had reduced antigenicity. DNA immunization in BALB/c mice revealed that wtHBsAg and vtHBsAg with T123N and K160N are able to induce antibodies to HBsAg (anti-HBs), whereas K1221 and A159G greatly impair the ability of HBsAg to trigger anti-HBs responses. The cellular immune response to the HBsAg aa 29-38 epitope was enhanced by the K160N substitution. Using replication competent clones of hepatitis B virus (HBV), T123N and A159G substitutions were shown to strongly reduce virion assembly. The amino acid substitution K160N appeared to compensate for the negative effect of All 59G on virion production. These results reveal complex effects of amino acid substitutions on biochemical properties of HBsAg, on antigenicity and immunogenicity, and on the replication of HBV.