We studied the transport of ¹⁵N from a soil compartment separated from a plant root compartment by a hydrophobic polytetrafluoroethylene (PTFE) membrane to plants in the presence and absence of arbuscular mycorrhizal fungi (AMF). We have previously shown that this type of membrane efficiently inhibits mass flow and diffusion of mobile ions in the soil solution in an abiotic system, but can be penetrated by the hyphae of mycorrhizal fungi. Mycorrhizal tomatoes (Lycopersicon esculentum) colonized by Glomus mosseae were grown at two N fertilizer concentrations in a root compartment. A PTFE membrane was placed between the root compartment and an adjoining soil compartment that was inaccessible to the roots but accessible to the AMF hyphae (hyphal compartment). Additional N was applied to the hyphal compartment using uniformly ¹⁵N‐labelled NH₄NO₃. There was a flux of ¹⁵N from the hyphal compartment to the plants even in the absence of mycorrhizal fungi. However, this flux was much higher in mycorrhizal plants, which had much higher N concentrations in their shoots and roots than did the non‐mycorrhizal control plants. This was particularly apparent when the root compartment had a low N fertilizer concentration. Of the total N content of mycorrhizal plants, c. 42 and 24% at the low and high N fertilizer concentrations, respectively, were estimated to originate from the hyphal compartment by transport through AMF hyphae. In the presence of mycorrhizal fungi, the flux of ¹⁵N was about three times higher than in their absence. The results show that AMF can access a soil compartment separated by a PTFE membrane, and can contribute substantially to N uptake by plants.