Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of -19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline-remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.When [methyl-(14)C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more (14)C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [(14)C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more (14)C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [(14)C]formate for about 18 hours in darkness, betaine was always the principal (14)C-labeled soluble metabolite. This (14)C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of (14)C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [(14)C]formate.These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.