Legionella pneumophila is a facultative intracellular human pathogen and an important cause of Legionnaires' disease, a severe form of pneumonia. Recently, we showed the presence of a putative twin-arginine translocation (Tat) pathway in L. pneumophila Philadelphia-1. This secretion pathway is used to transport completely folded proteins across the cytoplasmic membrane. The importance of the Tat pathway in L. pneumophila was investigated by constructing a tatB and a tatC mutant. Functionality of the Tat pathway was shown using a proven heterologous Tat substrate. It was shown that tatB and tatC are involved in intracellular replication in Acanthamoeba castellanii and differentiated U937 cells, and in biofilm forming ability. A putative Legionella Tat substrate was identified via 2D gel electrophoresis.