This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which selenocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and casein as a model protein for the GSHPx apo‐enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept selenocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl‐tRNA was converted to selenocysteyl‐tRNA by incubation with H2Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to selenocysteine residues by incubation with H2Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co‐ and/or post‐translational mechanisms.