A full-length transcript, Imt1, encoding myo-inositol O-methyltransferase (EC 2.1.1.X) from the halophyte Mesembryanthemum crystallinum was expressed in Escherichia coli. The enzyme, IMT1, uses S-adenosyl-L-methionine to methylate myo-inositol to form D-ononitol. IMT1 with a monomeric mass of 41,000 was isolated by ammonium sulfate fractionation, gel filtration and ion exchange chromatography to apparent purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified recombinant enzyme was identical to that encoded by the cDNA sequence. The apparent Km for S-adenosylmethionine was 0.18 mM with a Vmax of 1550 pkat/mg protein. The Km for myo-inositol was 1.32 mM. The reaction became substrate-inhibited by concentrations of S-adenosylmethionine greater than 0.5 mM. Inositol methyltransferase was competitively inhibited 50% with 0.01 mM S-adenosyl-homocysteine, while 1 mM homocysteine, homoserine, or adenosine did not inhibit. The enzyme exhibited a pH optimum of 7.8 and a temperature optimum of 37 degrees C. Activity of the isolated inositol methyltransferase was stable when stored at 4 degrees C.