RNAi is a powerful method for generating loss of function mutants, especially for targeting genes belonging to large gene families. We have recently shown that RNAi functions in the moss Physcomitrella patens. We obtained stable lines that show constitutive silencing of a nuclearly localized GFP:GUS fusion protein (NLS:GFP:GUS). However, lines that display silencing of the protein do not necessarily have reduced transcript levels. Therefore, a system has been developed that silences the NLS:GFP:GUS reporter construct at the same time as it silences a gene of interest. Gateway (Invitrogen) recombination cassettes were incorporated into these vectors to facilitate cloning of many different cDNA sequences. In addition, vectors were generated that contain genomic moss DNA sequence information to increase the production of stable moss lines. Transformation with these constructs results in strong silencing within 24 h and is stable for at least a month after transformation. FtsZ2-1, whose loss of function phenotype is known, was incorporated as a test case for analyzing phenotypes. One hundred per cent of regenerating colonies that have silenced GFP exhibit a loss of function FtsZ2-1 phenotype, validating the use of this system to assay phenotypes for plant genes of unknown function.