RNA segment 8 (NS) of influenza A virus encodes two proteins. The NS1 protein is translated from the unspliced primary mRNA transcript, whereas the second protein encoded by this segment, NS2/NEP, is translated from a spliced mRNA. Splicing of influenza NS1 mRNA is thought to be regulated so that the levels of NS2 spliced transcripts are approximately 10 % of total NS mRNA. Regulation of splicing of the NS1 mRNA has been studied at length, and a number of often-contradictory control mechanisms have been proposed. In this study, we used (32)P-labelled gene-specific primers to investigate influenza A NS1 mRNA splicing regulation. It was found that the efficiency of splicing of NS1 mRNA was maintained at similar levels in both virus infection and ribonucleoprotein-reconstitution assays, and NS2 mRNA comprised approximately 15 % of total NS mRNA in both assays. The effect of NS1 protein expression on the accumulation of viral NS2 mRNA and spliced cellular beta-globin mRNA was analysed, and it was found that NS1 protein expression reduced spliced beta-globin mRNA levels, but had no effect on the accumulation of NS2 mRNA. We conclude that the NS1 protein specifically inhibits the accumulation of cellular RNA polymerase II-driven mRNAs, but does not affect the splicing of its own viral NS1 mRNA.