The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.