To evaluate the bioavailability of selenium (Se) in high-Se yeast (SeY), the digestibility and chemical species of Se in SeY were investigated. Both Se and nitrogen in SeY were readily released into the soluble fraction through trypsin digestion. In a Sephadex G-25 gel chromatography of the trypsin digest of SeY, the range in which Se was eluted was coincident with the range in which peptide fragments were eluted. Se was distributed almost uniformly within the range and there was no fraction that contained Se in a specifically high amount. A proteolytic enzyme extract of SeY was found to contain Se as selenomethionine (74.8%), selenocystine (9.9%), selenite (5.1%) and as at least three unknown Se compounds (10.2%) when analyzed using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). These results indicate that Se in SeY is mainly present as selenomethionine non-specifically incorporated into peptide chains and is highly digestible. Accordingly, it is concluded that the bioavailability of Se in SeY is high.