Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.