A proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS was developed to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast. Capillary HPLC-inductively coupled plasma mass spectrometry (ICP-MS), employed in parallel to capHPLC-ESI MS, indicated the virtual independence of the ESI MS response of the peptide structure (in the elution range of 30-65% methanol), and hence, the use of ESI MS data to determine the SeCys/Cys and SeMet/Met substitution ratios. For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. The SeMet/Met and SeCys/Cys ratios were determined in a large number of peptides (57 and 26, respectively) issued from the tryptic digestion of 19 Se-containing proteins located in the gel by laser ablation-ICP MS imaging. The average Se/S substitution in methionine was 42.9. ±. 35.0 and was protein dependent with ratios ranging from 5 to 160 for individual peptides. The substitution of sulphur in cysteine (14.1. ±. 4.8%) in the cysteine-containing peptides was relatively similar (ratios from 9 to 23). Taking into account that the cysteine/methionine average ratio (2:1) in the yeast protein fraction, the study allowed the conclusion that 10-15% of selenium present in Se-enriched yeast is in the form of selenocysteine making up the mass balance of selenium species. Biological significance: For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. It was achieved using a proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS in order to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast.