In the course of studies on free amino acids in dried Shiitake [Lentinus edodes (Berk) Sing.], several unidentified ninhydrin-positive substances were obtained. Two of them were identified as nicotianine (N-(3-amino-3-carboxypropyl)-β-carboxypyridinium betaine) and L-cystathionine, and this paper describes their isolation and identification. Nicotianine, previously obtained from tobacco leaves, is the first reported pyridinium-containing amino acid isolated from fungi. Chromatographic determination showed the edible mushroom contains about 0.7 μmol of nicotianine per g of dry matter, with no quantitative differences between fresh and dried mushrooms, and the pileus contains a relatively larger amount than the stalk. Experiments on the nutritional effects of nicotianine are under way.