Expression profiling of fungal genes in the arbuscular mycorrhiza (AM) symbiosis has been based on studies of RNA extracted from fungal tissue or mycorrhizal roots, giving only a general picture of overall transcript levels in the targeted tissues. Information about the spatial distribution of transcripts within AM fungal structures during different developmental stages is essential to a better understanding of fungal activity in symbiotic interactions with host roots and to determine molecular events involved in establishment and functioning of the AM symbiosis. The obligate biotrophic nature of AM fungi is a challenge for developing new molecular methods to identify and localize their activity in situ. The direct fluorescent in situ (DIFIS) RT-PCR procedure described here represents a novel tool for spatial mapping of AM fungal gene expression simultaneously prior to root penetration, within fungal tissues in the host root and in the extraradical stage of fungal development. In order to enhance detection sensitivity of the in situ RT-PCR technique and enable localization of low abundance mRNA, we have adopted direct fluorescent labeling of primers for the amplification step to overcome the problem of low detection associated with digoxigenin or biotin-labeled primers and to avoid the multiplicity of steps associated with immunological detection. Signal detection has also been greatly improved by eliminating autofluorescence of AM fungal and root tissues using confocal microscopy.