Two overlapping DNA clones representing more than half of the Pichinde arenavirus S RNA segment were cloned into pBR322 and their nucleotide sequences were determined. The analyses predict that the viral nucleocapsid protein (N) is encoded in a reading frame in the viral complementary RNA sequence starting at viral S RNA nucleotide residue 84 from the 3' end and terminating with an opal codon at residues 1767-1769. The position of the termination codon has been confirmed by primer directed dideoxynucleotide sequencing. The N protein has a calculated size of 62,911 Da and a net positive charge of +9. Viral complementary 15 S mRNA that directs the synthesis of N protein and hybridizes to the predicted N gene DNA has been identified in infected cell extracts. A second nonoverlapping reading frame in the viral complementary sequence originates at nucleotide position 1827 and remains open for at least 71 amino acids (i.e., the extent of the second clone). A long stretch of hydrophobic amino acids is near the amino terminus of this predicted gene product. Between the two reading frames is a 60-nucleotide-long noncoding intergenic region. This nucleotide sequence can be arranged in hairpin configuration involving 14 G-C and 4 A-U base pairs. The possible function of this intergenic region in the regulation of transcription and/or translation is discussed.