A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the rapid determination of free ergosterol, ergosteryl esters, and ergocalciferol. The HPLC method was used to evaluate the distribution of ergosterol and ergosteryl esters in the different parts (stipe, pileus, and gills) of the agaric fungi, Agrocybe aegerita, Termitomyces albuminosus, and Lentinus edodes, and the relative changes of free and esterified ergosterols during the degradation of ergosterol in the comminuted fungal tissues. The results showed that total ergosterol levels and the relative abundances of free to esterified ergosterols were different among the various species and in the different parts of these agaric fungi. The results also indicated that ergosteryl esters were more stable than free ergosterol. While the content of free ergosterol markedly decreased, substantial amounts of ergosteryl esters remained for a long period, and even an increase in the contents of ergosteryl esters was also found in some comminuted fungal tissues. Therefore, it is possible that free ergosterol in the cell membrane of the dead fungal hyphae undergoes degradation or esterification, by which excess free ergosterol may be removed, and stored in cytosolic lipid particles. It is suggested that free ergosterol (not total ergosterol) should be used as a biomarker for fungal biomass.