The influenza virus NS1 protein is a unique posttranscriptional regulator that has two activities: inhibition of the nuclear export of poly A-containing mRNAs and inhibition of pre-mRNA splicing. Here we demonstrate that this protein binds to a specific region in one of the human spliceosomal snRNAs, U6 snRNA. Using U6 deletion mutations, we show that the binding of the NS1 protein requires both chains of a stem-bulge structure encompassing nucleotides 27-46 and nucleotides 83-101 of human U6 snRNA. A chemical modification/interference assay indicated that the primary binding site is centered around a purine-containing bulge in this stem-bulge structure. These results provide strong evidence that this postulated secondary structure in U6 snRNA actually exists. The NS1 protein also binds to a model U6-U4 snRNA complex, suggesting that the U6 stem-bulge comprising the NS1 protein binding site is also present in natural U6-U4 snRNA complexes. The U6 stem-bulge includes the U6 sequence that forms helix II with U2 snRNA during splicing, an interaction that is essential for mammalian splicing. We demonstrate that the NS1 protein blocks formation of the U6-U2 helix II both in a model system and during in vitro splicing. In addition, we show that the NS1 protein inhibits formation of U6-U4 snRNA complexes during in vitro splicing, presumably because the binding site of the NS1 protein includes the 3'-terminal region of U6 snRNA that has been shown to be important for the formation of U6-U4 complexes. We postulate that the inhibition of U6-U2 and U6-U4 snRNA complex formation is largely responsible for the inhibition of pre-mRNA splicing by the NS1 protein.