A fraction of the large surface protein (L) of duck hepatitis B virus (DHBV) is phosphorylated at serine or threonine residues (E. Grgacic & D. Anderson, Journal of Virology 68, 7344-7350, 1994). We now report the identification of phosphorylation sites in DHBV L protein. Using site-directed mutagenesis, we have identified serine-118 (S118) as the major phosphorylation site, accepting approximately 64% of the total phosphate groups incorporated in L, and resulting in retarded migration of phosphorylated L in SDS-PAGE. Proline-119 is indispensable for S118 phosphorylation. Mutation of other serine/threonine residues which are followed by prolines (T79, T89, S117 and T155) together with S118 further reduced phosphorylation to around 19% of wild-type. Non-equilibrium pH gel electrophoresis (NEPHGE) and SDS-PAGE of 33P-labelled L protein revealed two phosphorylated L species, while protein with the S118 to alanine mutation was detected as only one labelled species, consistent with multiple phosphorylations in wild-type L. Together, these results demonstrate that serine 118 is the major phosphorylation site for a proline-directed kinase, and that a proportion of L molecules are additionally phosphorylated at one of a number of secondary sites. DHBV mutants encoding L proteins with minimal phosphorylation (alanine mutants) or mimicking constitutive phosphorylation (aspartic acid mutants) remained infectious both in cell culture and in ducks, demonstrating that L phosphorylation may play only a minor role in DHBV replication.