The major selenium compound in an aqueous extract of the most popular mushroom in Eastern Asian countries, shiitake ( Lentinula edodes), fortified with selenium (Se) was identified by means of hyphenated techniques, i.e. HPLC-inductively coupled argon plasma mass spectrometry and HPLC-electrospray ionization mass spectrometry (HPLC-ICP MS and HPLC-ESI MS). Sixty-eight per cent of the total Se in the selenized shiitake was extracted with water, and 49.8% of the Se in the water extract was eluted in the high molecular mass fraction (>40,000 kDa) before incubation at 37 degrees C. After incubation, 40.6% of the Se in the water extract was eluted in a lower molecular mass fraction and the Se eluted in the high molecular mass fraction had decreased to 14.0%, suggesting that the major selenium compound in the water extract was initially in a form bound to macromolecule(s) and was then enzymatically liberated from the macromolecule(s). The retention time of the liberated selenium compound in HPLC-ICP MS matched that of selenomethionine (SeMet), and the masses of molecular and fragment ions detected by HPLC-ESI MS also suggested that the selenium compound was SeMet. The selenized shiitake accumulated Se as SeMet, and SeMet might be bound to the water extractable high molecular mass protein(s).