The replication activity of 2009 pandemic H1N1 influenza virus in human lung cells was evaluated in this study. Twenty-two surgically removed human lung tissue samples were infected ex vivo with pandemic H1N1 influenza virus (A/California/04/2009), seasonal human H1N1 influenza virus (A/Shantou/92/09), or a highly pathogenic H5N1 influenza virus (A/Vietnam/1194/04). Examination of nucleoprotein expression and viral RNA replication in the infected human lung tissue samples showed that whereas the replication of pandemic H1N1 influenza virus varied between tissue samples, overall this virus replicated more efficiently than seasonal H1N1 influenza virus but less efficiently than H5N1 influenza virus. Double-immunostaining for viral antigens and cellular markers indicated that pandemic H1N1 influenza virus replicates in type 2 alveolar epithelial cells.