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In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and localized in the cellular compartments.