The cap-dependent endonuclease activity of the influenza virus RNA-dependent RNA polymerase cleaves host mRNAs to produce capped RNA fragments for primers to initiate viral mRNA synthesis. The influenza virus type A (FluA) cap-dependent endonuclease preferentially recognizes cap1 structure (m(7)GpppNm). However, little is known about the substrate specificity of the influenza virus type B (FluB) endonuclease. Here, we determined the substrate specificity of the FluB polymerase using purified viral RNPs and [(32)P]-labeled polyribonucleotides containing a variety of cap structures, m(7)GpppGm, m(7)GpppG, and GpppG. We found that the FluA polymerase cleaves m(7)G-capped RNAs preferentially. In contrast, the FluB polymerase could efficiently cleave not only m(7)G-capped RNAs but also unmethylated GpppG-RNAs. To identify a key amino acid(s) related to the cap recognition specificity of the PB2 subunit, the transcription activity of FluB polymerases containing mutated cap-binding domains were examined by a mini-replicon assay system. In the case of FluA PB2, Phe323, His357, and Phe404, which stack the m(7)GTP, and Glu361 and Lys376, which make hydrogen bonds with guanine base, were essential for the transcription activity. In contrast, in the case of FluB PB2, the stacking interaction of Trp359 with guanine base and putative hydrogen bonds using Gln325 and Glu363 were enough for the transcription activity. Taken together also with the result of the cap-binding activity, we would propose that the cap recognition pocket of FluB PB2 does not have the specificity for m(7)G-cap structures, thus being more flexible to accept various cap structures than FluA PB2.