In the serum of hepatitis B virus (HBV)-infected patients, two different types of particles, a 42 nm virion and a 22 nm subviral particle, were identified. The envelope of both particles is composed of three proteins, the large (L), middle (M), and major/small (S) surface proteins but the ratio between these components varies in each. The M protein appears in a lesser amount than the S protein in both virion and subviral particles, although it is translated from the same subgenomic RNA, and this is due to its poor initiation context of translation. In addition, only the glycosylated form of M protein is secreted in contrast to both glycosylated and unglycosylated forms of L and S proteins that are secreted. To investigate the biogenesis of M protein, human hepatoma cells transfected with plasmids containing a mutated HBV DNA were used to produce a high amount of M protein. Electron microscopic observation revealed that despite a higher proportion of the M protein being found in the transfected cells, the secreted surface antigen particles possess similar size and density to 22 nm subviral particles. Detailed biochemical analyses showed the following. (1) The unglycosylated M protein was predominantly present in the microsomal fraction but not present in any other subcellular fractions. (2) The M protein formed 22-nm-like particles in the endoplasmic reticulum (ER) and was retained in the post-ER or pre-Golgi regions. (3) In addition to the complex glycosylated form of M protein, a high-mannose form of M protein could be secreted. (4) Normally, no unglycosylated M protein was secreted. However, glycosylation was not essential for M protein secretion since M protein deprived of glycosylation by tunicamycin treatment was detected in the medium. These findings suggest that (i) the M protein was probably translated and co-translocated into the ER and at least one site was glycosylated before leaving the ER resulting in no secretion of unglycosylated M protein, and (ii) the M protein had two secretion pathways, one through the conventional pathway and the other probably directly through the ER.