Arbuscular mycorrhizal (AM) fungi are important below-ground carbon (C) sinks that can be sensitive to increased nitrogen (N) availability. The abundance of AM fungi (AMF) was estimated in maple (Acer spp.) fine roots following more than a decade of experimental additions of N designed to simulate chronic atmospheric N deposition. Abundance of AMF was measured by staining and ocular estimation, as well as by analyzing for the AMF indicator fatty acid 16:1+5c in phospholipid (biomass indicator) and neutral lipid (lipid storage indicator) fractions. Arbuscular mycorrhizal fungal biomass, storage structures and lipid storage declined in response to N addition measured by both methods. This pattern was found when AM response was characterized as colonization intensity, on an areal basis and in proportion to maple above-ground biomass. The phospholipid fraction of the fatty acid 16:1+5c was positively correlated with total AMF colonization and the neutral lipid fraction with vesicle colonization. Decreased AMF abundance with simulated N deposition suggests reduced C allocation to these fungi or a direct soil N-mediated decline. The fatty acid (phospholipid and neutral lipid fractions) 16:1+5c was found to be a good indicator for AMF active biomass and stored energy, respectively.