Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown. The potential role of specific amino acid substitutions in the S protein from OBI in HBsAg production and excretion was examined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfected with plasmids containing the S gene of eight HBsAg(+) controls and 18 OBI clones. The intracellular (IC)/extracellular (EC) HBsAg production ratio was ∼1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strains clones: pattern 1, an IC/EC ratio of 1.0, was found in 5/18 OBI clones, pattern 2, detectable IC but low or undetectable EC HBsAg (IC/EC, 7.0 to 800), was found in 6/18 OBIs, and pattern 3, low or undetectable IC and EC HBsAg, was found in 7/18 clones. Intracellular immunofluorescence staining showed that in pattern 2, HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. The substitution M75T, Y100S, or P178R was present in 4/6 pattern 2 OBI clones. Site-directed-mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introduced into a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies expressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of two OBI strains may contribute significantly to the OBI phenotype.