The subcellular and developmental distribution of beta-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form beta-cyanoalanine and H(2)S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of beta-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.beta-Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total beta-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves.