The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein. (HEPATOLOGY 1998;27:1408-1414.) Woodchuck hepatitis virus (WHV) is a small enveloped DNA virus of the Hepadnaviridae family. The WHV genome encodes viral polymerase, core protein, X protein, and envelope proteins. Distinct polypeptides known as large (pre-S1), middle (pre-S2), and small (S) envelope proteins are independently translated from their own specific viral messenger RNA transcripts. Because the translation initiation codons Abbreviations: WHV, woodchuck hepatitis virus; ORF, open reading frame; DDPLL, group of five amino acid residues, Asp-Asp-Pro-Leu-Leu; DHBV, duck hepatitis B virus; GDPAL, group of five amino acids, Gly-Asp-Pro-Ala-Leu; CMV, cytomegalovirus.