La Crosse virus is a member of the Bunyavirus genus in the family Bunyaviridae, viruses with trisegmented RNA genomes of mostly negative polarity composed of large (L), medium (M), acid small (S) segments. The sequences of the La Crosse/original M and S RNA segments have been previously characterized. Using reverse transcriptase in conjunction with PCR amplification, we have obtained the nucleotide sequence of the L RNA segment, which encodes the viral polymerase in a single large open reading frame. Comparison of the amino acid sequence of the LAC L protein with the sequence of other polymerases from members of the Bunyaviridae, demonstrated the presence of several conserved motifs, some of which are characteristic of many polymerase proteins. A genetic tree comparing the available polymerase proteins of the Bunyaviridae provides insights into the phylogenetic relationships within this large family. Members of the genus Bunyavirus, which are mosquito-borne and infect mammals, have a closer relationship to the plant viruses represented by tomato spotted wilt virus (Tospovirus genus) than to viruses of other genera in the family Bunyaviridae. (C) 1995 Academic Press, Inc.