The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.