Agnoproteins, encoded by the 5'-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions. In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed. Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication. Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles. Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells. With proteins expressed in E. coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind. A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding. Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.