The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have 'rescued' the inactive PB1 protein.