A survival strategy operating in the absence of the host was shown in obligately biotrophic arbuscular mycorrhizal (AM) symbionts. When no host-derived signals from the surrounding environment were perceived by germinating spores, fungal hyphae underwent a programmed growth arrest and resource reallocation, allowing long-term maintenance of viability and host infection capability. The early stages of mycelial growth of AM fungi were studied by a combination of time-lapse and video-enhanced light microscopy, image analysis, and immunodetection, with the aim of acquiring knowledge of cell events leading to the arrest of mycelial growth. The time-course of growth arrest was resolved by precisely timing the growth rate and magnitude of the mycelium originating from individual spores of Glomus caledonium. Extensive mycelial growth was observed during the first 15 days; thereafter, fungal hyphae showed retraction of protoplasm from the tips, with formation of retraction septa separating viable from empty hyphal segments. This active process involved migration of nuclei and cellular organelles and appeared to be functional in the ability of the fungus to survive in the absence of a host. Immunodetection of cytoskeletal proteins, metabolic activity, and the retention of infectivity of germinated spores confirmed the developmental data. The highest amounts of tubulins were detected when hyphal growth had ceased but when retraction of protoplasm was most active. This was consistent with the role of the cytoskeleton during protoplasm retraction. Succinate dehydrogenase activity in hyphae proximal to the mother spore was still detectable in 6-month-old mycelium, which remained viable and able to form appressoria and produce symbiotic structures.