Two overlapping cDNA clones corresponding to the 5' region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an ambisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5' terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3' termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression.