β-Alanine (β–Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, β-Ala betaine is synthesized by S-adenosyl-l-methionine-dependent N-methylation of β-Ala via N-methyl β-Ala and N,N-dimethyl β-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of β-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating β-Ala, N-methyl β-Ala, and N,N-dimethyl β-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent Km of 45 μm S-adenosyl-l-methionine and substrate inhibition was observed above 200 μm. The apparent Km values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mm for β-Ala, N-methyl β-Ala, and N,N-dimethyl β-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.