A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)