The new world arenavirus Pichinde (PIC) is the basis of an accepted small animal model for human Lassa fever. PIC (Munchique strain) variant P2 is attenuated in guinea pigs, whereas variant P18 is extremely virulent. Previous sequence analysis of the S segments of these two viruses indicated a small number of possible virulence markers in the glycoprotein precursor (GPC) and nucleoprotein (NP) genes. In order to determine the role of these S segment genes in guinea pig virulence in this system, we have generated reassortant viruses. When tested in outbred guinea pigs, the reassortant containing the S segment from the virulent parent P18 (S18L2) caused significantly higher morbidity than the reciprocal reassortant. This increased morbidity was associated with higher viral titers in serum and spleen. However, the S18L2 reassortant was not as fully virulent in this system as the P18 parent, indicating a role for L segment genes in virulence.