The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1ω 5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating C-13 enrichment of 16:1ω 5 and compared it with C-13 enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [C-13] glucose. The 13 C enrichment of neutral lipid fatty acid 16:1ω 5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for C-13 in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1w5 than for the root specific neutral lipid fatty acid 18:2ω 6,9. We labeled plant assimilates by using (CO2)-C-13 in whole-plant experiments. The extraradical mycelium often was more enriched for C-13 than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between 13C enrichment in neutral lipid fatty acid 16:1ω 5 and total C-13 in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the C-13 enrichment of 16:1ω 5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.