An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.