In previous studies, human hepatitis viruses have been experimentally transmitted to New World monkeys of the genus Saguinus (tamarins). Recently, two Flaviviridae-like agents (GBV-A and GBV-B) were identified in tamarins that developed hepatitis following inoculation with serum of the 11th tamarin passage of a potentially new human hepatitis agent. However, it was not shown that these viruses originated from the initial inoculum. We here report the discovery of indigenous species-specific viruses related to GBV-A in several species of New World monkeys and suggest that GBV-A virus was fortuitously acquired during passage in tamarins. Sera or plasma from 98 wild-caught New World monkeys representing 10 different species was tested by RT-PCR with conserved degenerate primers to the 5' noncoding region of the genome. Viral sequences were identified in 33 animals and sequence analysis was performed on the amplicons. In addition, the genomic region corresponding to the putative NS3 RNA helicase of GBV-A was amplified from most positive animals and sequenced. We detected GBV-A-like viruses in 13 (35%) of 37 S. mystax, 7 (78%) of 9 S. nigricollis, 3 (25%) of 12 S. labiatus, 2 (50%) of 4 S. oedipus, 2 (100%) of 2 Callithrix jacchus, and 6 (50%) of 12 Aotus trivirgatus monkeys. Each positive animal was infected with a unique strain of the GBV-A-like viruses. Analysis of the 5' NC and NS3 helicase sequences revealed that these viruses could be classified into 5 major genetic groups with genetic distances equivalent to or greater than those found among major genetic groups of hepatitis C virus. Species-specific GBV-A-like viruses were found in S. mystax, S. nigricollis, S. oedipus, C. jacchus, and A. trivirgatus species. The viruses specific for S. nigricollis were closely related to GBV-A, suggesting that GBV-A was acquired by passage through this species during the initial transmission studies. The natural history of the GBV-A-like viruses was studied in serial serum samples from 9 S. mystax and 2 A. trivirgatus monkeys. Each animal was chronically infected and the viral strain did not vary during 9-27 months of follow-up. Finally, we demonstrated that four S. mystax were positive upon arrival to the United States from the country of origin. No apparent disease was associated with chronic infection of the GBV-A-like viruses. In conclusion, many New World monkeys are persistently infected with indigenous species-specific viruses that may represent a new genus within the virus family Flaviviridae.