The canonical pathway for isoleucine biosynthesis in plants begins with the conversion of threonine to 2-ketobutyrate by threonine deaminase (OMR1). However, demonstration of methionine gamma-lyase (MGL) activity in Arabidopsis (Arabidopsis thaliana) suggested that production of 2-ketobutyrate from methionine can also lead to isoleucine biosynthesis. Rescue of the isoleucine deficit in a threonine deaminase mutant by MGL overexpression, as well as decreased transcription of endogenous Arabidopsis MGL in a feedback-insensitive threonine deaminase mutant background, shows that these two enzymes have overlapping functions in amino acid biosynthesis. In mgl mutant flowers and seeds, methionine levels are significantly increased and incorporation of [(13)C]Met into isoleucine is decreased, but isoleucine levels are unaffected. Accumulation of free isoleucine and other branched-chain amino acids is greatly elevated in response to drought stress in Arabidopsis. Gene expression analyses, amino acid phenotypes, and labeled precursor feeding experiments demonstrate that MGL activity is up-regulated by osmotic stress but likely plays a less prominent role in isoleucine biosynthesis than threonine deaminase. The observation that MGL makes a significant contribution to methionine degradation, particularly in reproductive tissue, suggests practical applications for silencing the expression of MGL in crop plants and thereby increasing the abundance of methionine, a limiting essential amino acid.