Posttranscriptional gene silencing is a mechanism of suppressing gene expression in a sequence specific manner. This naturally occurring phenomenon of RNA interference (RNAi) has been used by researchers for gene function analysis in plants, fungi and animals. There is currently no report on RNAi establishment in mycorrhizal fungi. In order to obtain an insight into the in-vivo function of recently described genes coding for transporters and enzymes involved in N uptake and metabolism (Müller et al., 2007) from H. cylindrosporum, it is necessary to establish several methods for genetic analysis. After successfully expressing an EGFP in the mycelium of H.c. (Müller et al., 2006) we will present here our first assays on the establishment of the RNAi approach in H. cylindrosporum. In vitro the interfering agents is formed by annealing to the sense version of the gene an anti-sense version over a linder or spacer. The sense and anti-sense RNAs hybridize, the linker forms the characteristic stem-loop. The thus formed pre-miRNA is then exported from the nucleus into the cytoplasm to be cleaved by the enzyme Dicer. This enzyme initiates the RNAi-pathway, which is an innate cellular process. The dsRNA is cleaved into miRNAs, which are recognized by RISC. This effectively induces a loss of function and/or accompanying gain of function of another gene. The sense-linder-anti-sense fragment is inserted into a binary vector, after the introduction of a promoter-terminator set which allows it to function in the target fungus. On successful assemblage this will be a valuable tool to analyze the functioning and expression of transport proteins. Müller T. et al. (2007). Phytochemistry 68: 41-51 Müller T. et al. (2006). Mycorrhiza, 16: 437-442