We have investigated selenocysteine (2-amino-3-hydroselenopropionic acid) synthesis with cystathionine beta-synthase (EC 22.214.171.124) and cystathionine gamma-lyase (EC 126.96.36.199) of rat liver. When selenohomocysteine and serine were incubated with cystathionine beta-synthase, selenocystathionine was formed at a rate of 69% of that of cystathionine synthesis. Cystathionine gamma-lyase catalyzed alpha, gamma elimination of selenocystathionine to yield alpha-ketobutyrate, selenocysteine, and NH3. The reaction rate was about 3 times higher than that of cystathionine elimination. Cystathionine beta-synthase, however, did not catalyze direct formation of selenocysteine from serine and H2Se. Thus, selenocysteine is synthesized from selenohomocysteine and cystathionine beta-synthase and cystathionine gamma-lyase reactions. We confirmed this synthetic pathway also with a mixture of both enzymes and with a homogenate of rat liver.