The selenium concentration in Agaricus bisporus cultivated in growth compost irrigated with sodium selenite solution increased by 28- and 43-fold compared to the control mushroom irrigated solely with water. Selenium contents of mushroom proteins increased from 13.8 to 60.1 and 14.1 to 137μgSe/g in caps and stalks from control and selenised mushrooms, respectively. Selenocystine (SeCys; detected as [SeCys]2 dimer), selenomethionine (SeMet), and methyl-selenocysteine (MeSeCys) were separated, identified and quantified by liquid chromatography-electrospray ionisation-mass spectrometry from water solubilised and acetone precipitated proteins, and significant increases were observed for the selenised mushrooms. The maximum selenoamino acids concentration in caps and stalks of control/selenised mushrooms was 4.16/9.65μg/g dried weight (DW) for SeCys, 0.08/0.58μg/g DW for SeMet, and 0.031/0.10μg/g DW for MeSeCys, respectively. The most notable result was the much higher levels of SeCys accumulated by A. bisporus compared to SeMet and MeSeCys, for both control and selenised A. bisporus.