Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.