To determine whether the simple removal of any one amino acid was sufficient to extend chronological lifespan, we aged wild-type yeast in normal media, as well as four other media formulations, each lacking a randomly selected amino acid. We observed no lifespan extension for cells aged under these conditions. In addition, we found that genetic restriction of lysine, through deletion of the LYS2 gene, was similarly incapable of extending chronological lifespan. These data therefore indicate that the mere limitation of any particular amino acid is insufficent to extend chronological lifespan. Rather, the Meth-R-responsive pathway that confer extended lifespan in yeast do so in response to manipulations that specifically restrict methionine. Therefore, the benefit of Meth-R to yeast chronological lifespan is either reduced acid accumulation, resistance to acetic acid-induced death, or some combination thereof. To discriminate between these possibilities, we tested whether acid accumulation was affected in methioninerestricted 4-Aminohippuric Acid cultures by measuring the pH of normally aged cultures at varying intervals. We found that there was a direct correlation between age-related pH and lifespan, with cultures of long-lived cells genetically restricted for methionine demonstrating higher pH values. Measurements of WT cultures revealed a pH of 3.5 after 13 days of aging, whereas met15D cultures were less acidic even 12 days later. While this finding might superficially appear to be contrary to a previous report that pH is not altered between normal and methionine-restricted yeast during chronological aging, the authors of the aforementioned study Pramoxine hydrochloride assessed pH only at one timepoint, after 2 days of aging, and were therefore unable to report on subsequent pH changes. To determine whether genetic Meth-R might also render cells more tolerant to acetic acid-induced death, we assessed the survival of both wildtype and met15D cells after treatment with an extrinsic acetic acid source at a concentration similar to that typically achieved during chronological aging, as well as at higher concentrations, in order to offset the transient nature of the treatment.