The large envelope glycoprotein (L protein) of Hepatitis B virus (HBV) contains the preS1 domain, which is responsible for retention of the protein in the endoplasmic reticulum. To identify sequences of the preS1 domain involved in this phenomenon we constructed vaccinia virus-HBV recombinants containing the gene for L protein in which the preS1 coding sequence had been partially deleted. The retention of L protein in the endoplasmic reticulum was found to be mediated by a sequence contained within a region of 35 amino acids of the preS1 C-terminus, and not exclusively by amino acid sequences of the N-terminus of the preS1 domain as proposed by Kuroki et al. (Mol. Cell. Biol. 9, 4459-4466, 1989). Our finding could be explained by a specifically VV promoter sequence leading to exclusive synthesis of L or deleted (delta)L proteins, respectively. The ability of the coexpressed HBV S protein to facilitate export of the delta L proteins was demonstrated by coinfection experiments.