Several arenaviruses can cause hemorrhagic fever diseases (VHFs) in humans, the pathogenic mechanism of which is poorly understood due to their virulent nature and the lack of molecular clones. A safe, convenient, and economical small animal model of arenavirus hemorrhagic fever is based on guinea pigs infected by the arenavirus Pichinde (PICV). PICV does not cause disease in humans, but an adapted strain of PICV (P18) causes a disease in guinea pigs that mimics arenavirus hemorrhagic fever in humans in many aspects, while a low-passaged strain (P2) remains avirulent in infected animals. In order to identify the virulence determinants within the PICV genome, we developed the molecular clones for both the avirulent P2 and virulent P18 viruses. Recombinant viruses were generated by transfecting plasmids that contain the antigenomic L and S RNA segments of PICV under the control of the T7 promoter into BSRT7-5 cells, which constitutively express T7 RNA polymerase. By analyzing viral growth kinetics in vitro and virulence in vivo, we show that the recombinant viruses accurately recapitulate the replication and virulence natures of their respective parental viruses. Both parental and recombinant virulent viruses led to high levels of viremia and titers in different organs of the infected animals, whereas the avirulent viruses were effectively controlled and cleared by the hosts. These novel infectious clones for the PICV provide essential tools to identify the virulence factors that are responsible for the severe VHF-like disease in infected animals.Arenaviruses are enveloped RNA viruses with single-stranded ambisense RNA genomes (6). The genomes of these viruses consist of two RNA segments, the large (L) segment of ϳ7.2 kb and the small (S) segment of ϳ3.4 kb (6). The L RNA segment encodes the viral polymerase L protein in a negative orientation and a small multifunctional Z protein in a positive sense. The S RNA segment encodes the nucleoprotein NP in a negative orientation and the envelope glycoprotein precursor GPC in a positive sense (6). The terminal 19 nucleotides (nt) from both ends of the segments are imperfectly complementary to each other and are predicted to form the panhandle structures that serve as the cis-acting elements required for viral RNA transcription and replication (15,23,24,33). A unique feature of the arenavirus genomic RNAs is the noncoding intergenic regions located between the two open reading frames (6). They range from 59 to 217 nt in length and are predicted to form one to three energetically stable stem-loop structures (6, 40) that are proposed to contribute to transcriptional termination. Currently, the only available molecular clone of arenavirus was developed for the prototype lymphocytic choriomeningitis virus (LCMV) (13,38). This system has served as an invaluable tool to study the biological functions of arenavirus proteins and RNA elements.