The use of slower acid-producing starter bacteria for the production of lower fat Cheddar cheese has lead to milder flavor Cheddar cheeses that lack intense Cheddar notes. The metabolism of methionine leads to the production of methanethiol, which is one of the desirable Cheddar cheese flavor compounds. The influence of NaCl and reduced pH was determined for aminopeptidase, lipase/ esterase, and methanethiol-producing capability in selected lactic acid bacteria and brevibacteria in simulated cheese-like conditions. The activity of each enzyme decreased with NaCl addition and pH reduction to approximate a Cheddar cheese environment (5% NaCl and pH 5.2). The mechanism for methanethiol production by the starter and adjunct bacteria was also investigated. Different enzyme systems were found to be responsible for methanethiol production in starter lactococci, lactobacilli, and brevibacteria. In the lactococci, enzymes that acted primarily on cystathionine were responsible for methanethiol production from methionine. Lactobacilli also contained cystathionine-degrading enzymes, but these enzymes have properties different from the lactococcal enzymes. Brevibacterium linensBL2 lacked cystathionine-degrading enzymes, but was capable of the direct conversion of methionine to methanethiol. L-Methionine ��-lyase from B. linens BL2 was purified to homogeneity, and was found to catalyze the ��, �� elimination of methionine resulting in the production of methanethiol, ��-ketobutyrate, and ammonia. Characterization of the pure enzyme demonstrated that it is pyridoxal phosphate dependent, which is active at salt and pH conditions existing in ripening Cheddar cheese. The addition of either B. linens BL2 or L-methionine ��-lyase to aseptic cheese curd slurries increased methanethiol and total volatile sulfur compound production. In an attempt to increase methanethiol production and Cheddar cheese flavor in reduced-fat Cheddar cheese, B. linens BL2 was added as a starter adjunct to 60% reduced-fat cheese. Sensory evaluation of the cheese indicated that B. linens BL2 improved the flavor of 60% reduced-fat Cheddar cheese. This suggests that the addition of B. linens BL2 is an alternative to the addition of lactic acid bacteria to improve Cheddar cheese flavor via the metabolism of methionine.