Guanarito virus (the etiologic agent of Venezuelan hemorrhagic fever, VHF) and Pirital virus coexist in the region of Venezuela in which Venezuelan hemorrhagic fever is endemic. The purpose of this study was to extend our knowledge of the evolutionary relationship between these two arenaviruses. We determined that the large genomic segments of Guanarito virus and Pirital virus are similar in size and identical in structural organization to the large genomic segments of other South American arenaviruses. For example, the Z proteins and RNA-dependent RNA polymerases of Guanarito virus and Pirital virus are encoded in nonoverlapping open reading frames of opposite polarities. Phylogenetic analyses of Z protein gene nucleotide sequences and RNA-dependent RNA polymerase gene nucleotide sequences grouped Pirital virus with Pichindé virus (a South American arenavirus which, like Pirital virus, does not appear to be pathogenic for humans) and placed the Pirital-Pichindé lineage in a sister relationship to a lineage represented by Guanarito virus and the three other arenaviruses known to cause hemorrhagic fever in South America. These results are concordant with the results of studies on the phylogeny of the arenavirus small genomic segment. Thus, the exchange of genomic elements between Guanarito virus and Pirital virus via recombination or reassortment likely did not contribute to the emergence of Venezuelan hemorrhagic fever.