The prevalence in the population of hepatitis B virus (HBV) surface antigen (HBsAg) variants that may impair diagnosis, or allow the virus to escape vaccine-induced immunity or passive immunoglobulin therapy is unknown. A genome fragment encoding HBsAg amino acids 112-212 was amplified and sequenced from the sera of 272 unselected DNA-positive, HBV-chronic carriers from Spain. The genotype and the HBsAg subtype were predicted from the sequences. Analysis of amino-acid positions 112-157 revealed single or multiple substitutions in 39% of the carriers studied. Mutations were not detected for residues 121, 135, 137, 139, 140, 141, 142, 146, 147, 148, 149, 151, 152, 153, 155, 156, and 157. Substitutions reported previously to be in association with failures of diagnostic tests or with vaccine or immunoglobulin therapy escape were found in 12.5%, 6.6%, and 9.2% of carriers, respectively. Met133Thr (2.2%); Gln129His, Met133Ile, Phe/Tyr134Asn (1.8%); Phe/Tyr134Leu, Gly145Ala (1.5%), and Pro120Thr (1.1%) were the most frequent. Other substitutions, including Gly145Arg (0.4%), were found at a frequency of less than 1%. Samples containing HBV mutants were tested with three commercial assays for HBsAg screening. Almost all the mutants reacted to the upper cut-off values of the assays, but six samples with weak reactivity with one or more of the methods were also found. Thus, HBV mutants with a potential impact on clinical and public health issues are moderately frequent among chronic carriers from Spain, although their influence on the performance of diagnostic tests seems to be slight.