S-Adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT; EC 2.1.1.103) catalyzes the key step in choline (Cho) biosynthesis, the N-methylation of phosphoethanolamine. Cho is a vital precursor of the membrane phospholipid phosphatidylcholine, which accounts for 40 to 60% of lipids in nonplastid plant membranes. Certain plants use Cho to produce the osmoprotectant glycine betaine, which confers resistance to salinity, drought, and other stresses. An Arabidopsis mutant, t365, in which the PEAMT gene is silenced, was identified using a new sense/antisense RNA expression system. t365 mutant plants displayed multiple morphological phenotypes, including pale-green leaves, early senescence, and temperature-sensitive male sterility. Moreover, t365 mutant plants produced much less Cho and were hypersensitive to salinity. These results demonstrate that Cho biosynthesis not only plays an important role in plant growth and development but also contributes to tolerance to environmental stresses. The temperature-sensitive male sterility caused by PEAMT silencing may have a potential application in agriculture for engineering temperature-sensitive male sterility in important crop plants.