We developed a method for separating physiologically active intraradical hyphae of arbuscular mycorrhizal (AM) fungi from mycorrhizal roots, allowing the hyphae to be used for physiological and biochemical experiments. In the present study, the phosphate efflux from the intraradical hyphae in vitro was examined in relation to hyphal phosphatase activity. Onion seedlings (Allium cepa) were planted in the soil inoculated with Gigaspora margarita. Six weeks after transplanting, the intraradical hyphae were isolated from the mycorrhizal roots using plant cell-wall digestion enzymes. The hyphae were incubated briefly at 25 °C in a buffer solution (50 mM Tris/HCl, pH 7.4), then incubated for 2 h and gently shaken with various inhibitors. Phosphate efflux, the amount of phosphate released to the buffer, was analysed by EnzChek phosphate assay kit. Hyphal phosphatase activity was stained histochemically and the proportion of phosphatase-active arbuscules was examined for each inhibitor. Phosphate effluxes were to some degree reduced by all inhibitors used, while the phosphatase inhibitor, BeSO4, greatly reduced the efflux. The degree of inhibition in the arbuscular phosphatase by each chemical was closely correlated to the decrease in the phosphate efflux. These results suggest that hyphal phosphatase may be partially involved in the phosphate efflux process from intraradical hyphae.