The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5′ end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual and mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.