The RNA promoters of the genome and antigenome of Newcastle disease virus (NDV) were studied by mutational analysis of their 3' terminal ends. Similarly to other paramyxoviruses, NDV RNA replication follows the rule of six, and the genomic and antigenomic promoters require two discontinuous regions: conserved region I (first 18 nucleotides) and conserved region II (nucleotides 73-90). Proper spacing between those regions and the phase of six in region II is critical for efficient RNA promoter activity. As expected, the gene start signal at the 3' end of the NDV genome was required for mRNA transcription, but not for RNA replication. Surprisingly, mutation of the polyadenylation signal in the 5' end did not affect gene expression transcription. Although the conserved region I of NDV (avulavirus) promoter appears to be more similar to that of Sendai virus (SeV) (respirovirus), conserved region II is analogous to that of rubulaviruses.