We have been studying defective interfering (DI) genomes of the RNA enveloped virus Sindbis virus. Deletion mapping of a DI cDNA demonstrated that only sequences at the 3' and 5' termini of the genome are required for the DI RNA to be biologically active. We constructed a series of cDNAs that transcribe DI RNAs differing only in 5'-terminal sequences. Two of the 5' termini identical to ones found in naturally occurring DI RNAs are the 5' terminus of the virion RNA (DI-549) and the first 142 nucleotides from the 5' terminus of the subgenomic 26S mRNA attached to the 5' terminus of the virion RNA (DI-15). The latter has a 42-nucleotide deletion from nucleotides 25 to 66 in the 26S RNA sequence. These DI RNA transcripts were biologically active, but one (DI-526) which did not have the 42-nucleotide deletion of DI-15 was not replicated. The DI RNA isolated after the presumed amplification of the DI-526 transcript had deleted the first 54 nucleotides of the 26S RNA sequences. The 5' terminus of Sindbis virion RNA contains a stem and loop region that is conserved among alphaviruses. An 11-nucleotide deletion in DI-549 that disrupted this stem and loop rendered this DI RNA inactive. In contrast, this same deletion in DI-15 and one that removed an additional 100 nucleotides of the virion 5' terminus did not prevent its amplification. We did not detect by computer analysis any common secondary structures among the biologically active DI RNAs that distinguished them from those RNAs that were not amplified. Our results support the conclusion that tertiary structure or the ability of the RNA to adapt its structure upon interaction with protein is important in the recognition process.