Abstract A sequential sample preparation process was developed for the speciation analysis for Se-enriched edible mushroom, Agaricus bisporus containing 110.2 μg of total Se/g sample. Five different sample extraction methods were compared and the most efficient method (a three-step process involving the use of water extraction and two proteolytic enzymes — pepsin and trypsin) proved to be the most suitable for extracting selenium, with an extraction efficiency of 75%. As the analogues of these enzymes play an important role in human digestion the bioavailability of the selenium present in the sample was estimated. Selenocystine (SeCys 2) and Se(IV) were detected in considerable amounts (27.7 μg Se/g sample and 46.4 μg Se/g sample, respectively). For the quality control of peak identification a spiking procedure was developed, using a low selenium mushroom containing 4.3 μg of total Se/g sample. During the analysis with HPLC-HHPN (Hydraulic High Pressure Nebuliser)-AFS complicated background effects and matrix problems were observed: stable and reproducible signals generated by the low selenium mushroom and the compounds used in sample preparation were detected. The three-step sample preparation method connected with the HPLC-HHPN-AFS system proved to be applicable for the speciation analysis of the Se-enriched Agaricus bisporus.