We have analyzed the role of terminal sequences of a defective interfering (DI) particle RNA of vesicular stomatitis virus (VSV) in replication. A series of internal deletion mutants of DI cDNA was generated to obtain DI genomic RNAs that differed from one another by the presence of different lengths of 3'-terminal and/or 5'-terminal sequences. Analyses of the mutant. RNAs for their ability to replicate in cells transfected with the corresponding plasmids suggested that distinct regions at the termini of DI RNA are important for RNA replication. Region I, encompassing nucleotides 1-24, is absolutely required for replication since DI RNA genomes lacking any part of this region failed to replicate. Region II, spanning nucleotides 25-45, is not essential for replication but it functions as an enhancer of replication in that the presence of these specific sequences confers high efficiency of replication to the template. Deleting these specific sequences from both termini of DI RNA but maintaining the length of terminal complementarity as seen in wild-type DI RNA resulted in a template that replicated poorly (about 20-fold less efficiently). Furthermore, insertion or substitution of these sequences into the 3'-terminus of a VSV minigenome resulted in a template that replicated more efficiently (at least 4-fold to as high as 15-fold) than the parental minigenome. These results strongly support the conclusion that the presence of specific sequences rather than the extent of complementarity at the termini of DI RNA is a major determinant of the efficiency of replication. The presence of the specific sequences at the 3'-terminus of both genomic and antigenomic DI RNAs may explain in part the replicative dominance of DI RNA over the full-length VSV genome which contains these sequences only at the 3'-terminus of the antigenome.