In infected liver tissue three major and several minor duck hepatitis B virus (DHBV) envelope proteins are detectable by immunoblotting. Translation initiation at the second and the most distal internal ATG codon of the Pre-S/S gene is known to lead to synthesis of two major envelope proteins (P36 and P17) whereas the origin of a further major (P28) and other minor envelope proteins is not clear. Therefore, it was investigated whether translation initiation at pre-S ATGs leads to synthesis of these other envelope proteins and, if yes, whether they are components of viral particles and essential for infectivity. Each of the five ATG codons of the pre-S region of an infectious Chinese DHBV genome (DHBV26) was mutated separated by oligonucleotide-directed mutagenesis (point mutations or deletions) and the function of the corresponding mutant viruses were tested in vitro and in vivo. Immunoblot analysis of liver cell extracts or of extracts from hepatoma cells transfected with the DHBV genomes showed expression of minor pre-S proteins of about 35, 33, and 30 kDa. These proteins were not expressed when ATG codons at nucleotide positions 825, 882, and 957, respectively, were mutated. None of the ATG mutations abolished expression of the major P28 pre-S protein. In cell culture supernatants the minor pre-S proteins P35, P33, and P30 were identified as components of viral particles. With the exception of the DHBV genome containing the mutated ATG codon 801 (translation initiation codon for the major P36 pre-S protein) all forementioned DHBV mutant genomes were infectious. These data demonstrate that minor pre-S proteins are initiated at internal AUGs of the pre-S gene and are components of viral particles but are not essential for infectivity. In contrast to previously published speculations, the results also indicate that the major pre-S protein P28 is not initiated at AUG codon 957 but probably produced by proteolysis from larger pre-S proteins.