Glucosinolates are defensive compounds found in several plant families. We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles. In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST showing high sequence homology to one TMT isoform. The genomic clone of cTMT1 was subsequently amplified by PCR. Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca. 25 kDa), but differing in 13 residues. The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O- or S-methyltransferases. A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs. Southern analysis indicated single copy for each TMT gene. The two cDNAs were expressed in Escherichia coli. The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme. These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs. The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates. The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.