In yeast, the assignment of the threonine synthetase activity to the THR4 gene has been inferred from different data, but never really proved enzymatically. In this work, an assay system for threonine synthetase activity in yeast crude extract is reported. The method is based on the quantification by reverse-phase high-performance liquid chromatography, of the threonine formed from O-phosphohomoserine. Using this method we have determined that this activity depends on the presence in the cell of an active form of the THR4 gene, thus demonstrating the univocal relationship between them.