That the hemagglutinin (HA) of reovirus, encoded in the SI gene, determines the central nervous system (eNS) cell tropism of reovirus types I and 3 was shown using recombinant clones containing nine genes from one serotype and the SI gene from the other. Clone I.HA3 contains nine genes from type I and the SI gene from type 3; 3.HA1 is the reciprocal clone. Type 3 and I.HA3 cause a fatal encephalitis in newborn mice with neuronal destruction but no ependymal cell damage, whereas type I and 3.HA1 cause a nonfatal ependymal infection but no neuronal damage. Immunofluorescent studies showed no viral antigen in ependymal cells of mice infected with type 3 or I.HA3 or in neuronal cells of mice infected with type I or 3.HA1. With type 3 or clones containing the type 3 HA, maximal brain titers were 10¹⁰ plaque-forming units; maximal titers were 10⁸ plaque-forming units for type I or clones containing the type I HA. This pattern of reovirus virulence for CNS probably relates to the specific interaction of viral HA with neuronal or ependymal surface receptors.