A rapid and reliable thin-layer chromatographic (TLC) method for the quantitative analysis of ergothioneine in plasma and in various organs is presented. After extraction from biological materials with acetone/methanol (1 : 1), ergothioneine was separated by TLC and the spot corresponding to the compound with Rf 0.29 was quantified by scanning with a densitometer at 256 nm. The sensitivity of the assay was 0.05 μg/20μl of liver extract. This procedure should be useful in pharmacokinetic studies of ergothioneine in relation to its physiological role.