Influenza is generally regarded as an infection of the respiratory tract; however, neurological involvement is a well-recognized, although uncommon, complication of influenza A virus infection. The authors previously described the development of a rat model for studying influenza virus infection of the central nervous system (CNS). This model was used here to study the role of virus genes in virus replication and spread in brain. In the present work, an infectious cDNA clone of the neurotoxic WSN strain of influenza virus (rWSN) was altered by site-directed mutagenesis at five loci that corresponded to changes previously shown to confer temperature sensitivity and attenuation of the A/Ann Arbor/6/60 strain (PB1Delta 391, PB1Delta 581, and PB1Delta 661; PB2Delta 265, and NPDelta 34). Whereas rWSN and its mutated derivative (mu-rWSN) replicated equally well in MDCK cells at 37 degrees C (the body temperature of rats), rWSN grew to higher titers and infection was more widespread compared to mu-rWSN in rat brain. These results demonstrate that the five mutations that confer attenuation of the A/Ann Arbor/6/60 influenza virus strain for the respiratory system also confer attenuation for the central nervous system. Further in vivo and in vitro examination of these five mutations, both individually and in combination, will likely provide important information on the role of specific virus genes in virulence and pathogenesis.