Four DNA fragments that could rescue the mutations of four Met(-) mutants were cloned from Thermus thermophilus HB27 and their complete nucleotide sequences were determined. Two of the four fragments respectively contained the greater parts of the metF and metH genes, the predicted amino acid sequences of which showed identities of 30.8% and 32.7% with 5,10-methylenetetrahydrofolate reductase (EC 1.7.99.5) and vitamin B-12-dependent homocysteine transmethylase (EC 2.1.1.13) of Escherichia coil. The other two DNA fragments, which overlapped one another, contained two open reading frames whose predicted amino acid sequences were respectively similar to those of O-acetylhomoserine sulfhydrylase (EC 4.2.99.10, the product of the MET17 gene) and homoserine O-acetyltransferase (EC 2.3.1.31, the product of the MET2 gene) of Saccharomyces cerevisiae. The metF, metH, MET2, and MET17 genes of T. thermophilus were disrupted by introducing the heat-stable kanamycin nucleotidyltransferase gene into the genome. Each transformant showed methionine auxotrophy. Both the MET2- and MET17-disrupted mutants could grow in a minimal medium containing homocysteine but not in the same medium containing succinylhomoserine or cystathionine. In contrast, the metF- and metH-disrupted mutants could not grow in the minimal medium containing homocysteine. These results suggest that in T. thermophilus, homoserine is directly converted to homocysteine via O-acetylhomoserine and that homocysteine is methylated to synthesize methionine.