We have determined the nucleotide sequence of sacbrood virus (SBV), which causes a fatal infection of honey bee larvae. The genomic RNA of SBV is longer than that of typical mammalian picornaviruses (8832 nucleotides) and contains a single, large open reading frame (179-8752) encoding a polyprotein of 2858 amino acids. Sequence comparison with other virus polyproteins revealed regions of similarity to characterized helicase, protease and RNA-dependent RNA polymerase domains; structural genes were located at the 5' terminus with non-structural genes at the 3' end. Picornavirus-like agents of insects have two distinct genomic organizations; some resemble mammalian picornaviruses with structural genes at the 5' end and non-structural genes at the 3' end, and others resemble caliciviruses in which this order is reversed; SBV thus belongs to the former type. Sequence comparison suggested that SBV is distantly related to infectious flacherie virus (IFV) of the silk worm, which possesses an RNA of similar size and gene order.