The complete RNA sequence of the L protein gene of lymphocytic choriomeningitis virus (LCMV) is presented. It is the first L protein sequence to be obtained for the Arenaviridae, a family of single-stranded RNA viruses which includes Lassa fever virus, and the Tacaribe complex viruses such as Pichinde and the Argentine and Bolivian hemorrhagic fever viruses. It is the largest open reading frame on the L RNA spanning 6633 nucleotides and coding for a 2210 amino acid protein with a calculated molecular weight of 254,529. Antipeptide sera identify a gene product encoded on the L RNA: it has a mass of approximately 200,000 Da and is found in virions and ribonucleoprotein complexes from infected cells (M. Singh, F. Fuller-Pace, M. J. Buchmeier, and P. J. Southern, 1987, Virology, 161, 448-456). Mutations mapped to the L gene affect plaque morphology (Kirk et al., 1980), the lethality of a virulent LCMV strain on guinea pigs (Y. Riviere, R. Ahmed, P. J. Southern, M. J. Buchmeier, and M. B. A. Oldstone, 1985, J. Virol., 55, 704-709), and the ability of a variant strain of LCMV to suppress the cytotoxic T-cell response and initiate persistent infection (M. Salvato, E. Shimomaye, P. Southern, and M. B. A. Oldstone, 1988, Virology, 164, 517-522; Ahmed et al., 1988). All of these phenotypes indicate that the viral genes on the L strand are critical elements controlling virus replication and the pattern of LCMV infection. The L gene sequence encodes a viral polymerase although this protein bears little resemblance to the published sequences of other RNA virus polymerases. Therefore the LCMV polymerase likely represents a distinct category of viral transcriptase.