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The nucleotide sequence of a portion of the nucleocapsid (N) gene of the Guanarito virus prototype strain (INH-95551) has been determined. It was obtained by direct RNA and polymerase chain reaction (PCR) fragment sequencing of the 3′ end of the small (S) RNA fragment. A comparison of this 782-nucleotide segment was done with the known homologous gene sequences of five other arenaviruses: Junin, Machupo, Tacaribe, Pichinde, and lymphocytic choriomeningitis (LCM). Phylogenetic analysis of the N gene open reading frame showed that Guanarito virus is genetically distinct from other members of the Arenavirus family, with 32% nucleotide sequence divergence from Junin, 30% from Machupo, 32% from Tacaribe, 41% from Pichinde, and 45% from LCM. Comparison of amino acids encoded by this sequence region indicated a probable antigenic domain (amino acids 55–63) shared among all arenaviruses studied to date. Along with its host restriction and focal distribution, our data support the hypothesis that this virus has been evolving independently in its endemic focus, for some time.