Abstract Complete double-stranded DNA copies of the La Crosse virus (LAC) S genome have been synthesized and cloned into plasmid pBR322. The cloned genome was characterized and sequenced. The LAC S genome consisted of 981 nucleotides and contained two overlapping open reading frames. The first reading frame begins at nucleotide 82 and encodes a protein of 235 amino acids. A polypeptide of 92 amino acids can be translated in a +1 reading frame 16 nucleotides downstream from the start of the first reading frame. This second reading frame is initiated with two AUG codons followed by the serine codon UCG, the same serine codon which immediately follows the AUG of the first reading frame.