Natural mosses have been employed as reactive and accumulative indicators of atmospheric pollutants. Using the denitrifier method, the concentration, δ¹⁵N and δ¹⁸O of moss nitrate (NO3−) were measured to elucidate the sources of NO3− trapped in natural mosses. Oven drying at 55–70 °C, not lyophilization, was recommended to dry mosses for NO3− analyses. An investigation from urban to mountain sites in western Tokyo suggested that moss [NO3−] can respond to NO3− availability in different habitats. NO3− in terricolous mosses showed isotopic ratios as close to those of soil NO3−, reflecting the utilization of soil NO3−. Isotopic signatures of NO3−in corticolous and epilithic mosses elucidated atmospheric NO3− sources and strength from the urban (vehicle NO_x emission) to mountain area (wet-deposition NO3−). However, mechanisms and isotopic effects of moss NO3− utilization must be further verified to enable the application of moss NO3− isotopes for source identification.