Thiazolidine-4-carboxylic acid (TCA, thioproline) is a condensation product of cysteine and formaldehyde and an effective nitrite trapping agent in the human body that may block endogenous formation of carcinogenic N-nitroso compounds. This study investigated TCA levels in five edible mushrooms. The amounts of TCA in dried shiitake mushroom (Lentinus edodes) were 134 ± 137 μg (uncooked) and 843 ± 427 μg per 100 g (dry weight basis) when boiled, respectively. Uncooked raw shiitake mushroom contained no detectable TCA, but 406 ± 248 μg/100 g (dry weight basis) was formed during boiling. This TCA formation was effectively inhibited by addition of N-ethylmaleimide, an SH-trapping reagent. Dried shiitake samples soaked in water contain a specific system for generating formaldehyde via the formation of lenthionine from lentinic acid. The contents of TCA in other raw edible fungi examined (Agaricus bisporus, Collybia velutipes, Lyophyllum aggregatum, Tricholoma matsutake) were <3.8 ppm and with formaldehyde concentrations similar to that of raw shiitake mushroom.