To better understand the mechanisms by which neurotropic viruses invade peripheral nerve pathways and produce CNS disease, we defined the type 3 (T3) reovirus genes that are determinants of the capacity of reovirus T3 strain Dearing (T3D) and T3 clone 9 (C9) to infect the spinal cord and kill mice after hindlimb injection. T3D and C9 viruses are both highly virulent (LD(50) < 10(1) PFU) after intracranial injection of neonatal mice. However, C9 is significantly more lethal than T3D after either intramuscular injection (LD(50) < 10(1) vs LD(50) 10(4) PFU) or peroral injection (LD(50) 10(3.4) vs LD(50) > 10(8.3) PFU). Using reassortant viruses containing different combinations of genes derived from T3D and C9, we found that the S1 gene, encoding the cell attachment protein sigma 1 and the nonstructural protein sigma 1s, and the L3 gene, encoding the core shell protein lambda 1 were the primary determinants of lethality after intramuscular injection. The L3 gene and the L2 gene encoding spike protein, lambda 2, determined differences in spinal cord titer after intramuscular injection. A C9 x T3D mono-reassortant containing all T3D genes except for the C9-derived L3 was lethal after peroral injection. These studies indicate that the S1, L2, and L3 genes all play a potential role in neuroinvasiveness and provide the first identification of a role in pathogenesis for the L3 gene.