Nuclear distribution within the extra-radical fungal structures and during spore production in the arbuscular mycorrhizae fungus Glomus intraradices was examined using an in vitro monoxenic culture system. A di-compartmental monoxenic culture system was modified using a nitrocellulose membrane and a coverglass slip for detailed observations. Nuclear distribution was observed using the fluorescent DNA binding probes SYBR Green I and DAPI. Both septate and non-septate mycelial regions were observed, but cytoplasmic contents were only found within non-septate mycelia. Nuclear fluorescent staining revealed that the non-septate hyphal region contained nuclei only with cytoplasm, and that nuclear distribution was limited by septa. Swollen hyphal bodies were often associated with septate and empty-looking hyphae. Cytoplasmic contents filled the swollen hyphal body from the non-septate hyphal region following removal of the septa. As a consequence, the swollen body developed into a new spore. These observations provide understanding about the distribution of AM fungal nuclei within extra-radical mycelia and during spore formation. The results suggest a mechanism by which the development of a cytoplasm-containing mycelium is controlled by the formation or removal of septa to efficiently maintain and proliferate essential contents. This mechanism may provide a survival strategy to the fungus.