Several New World (Tacaribe complex) arenaviruses (Arenaviridae) are known to cause severe hemorrhagic disease in humans. Phylogenetic reconstruction of the Tacaribe complex arenaviruses previously has been limited by the relative scarcity of sequence data for arenavirus genomes. In the present study, oligonucleotide primers were designed based on conserved regions of the nucleocapsid (N) protein gene and then used to amplify, by reverse transcription--polymerase chain reaction, a 613-to 649-nucleotide region of the N gene of all known Tacaribe complex arenaviruses. This has allowed completion of the first detailed genetic characterization and phylogenetic analysis of all known members of the Tacaribe complex. These viruses formed three lineages. Lineage A contained Flexal, Parana, Pichinde, and Tamiami viruses; lineage B contained Amapari, Guanarito (GUA), Junin (JUN), Machupo (MAC), Sabia (SAB), and Tacaribe viruses. Latino and Oliveros viruses occupied lineage C. The highly pathogenic Tacaribe complex arenaviruses (GUA, JUN, MAC, SAB) were all members of lineage B, suggesting the possibility that the highly pathogenic phenotype is the result of evolutionary radiation from a common ancestor. The approach described here provides a rapid method for characterization of novel Tacaribe complex arenaviruses and may provide clues as to their potential public health importance.