Long-lived Ames dwarf mice lack growth hormone, prolactin, and thyroid stimulating hormone. Additionally the dwarf mice have enzyme activities and levels that combat oxidative stress more efficiently than those of normal mice. We have shown that methionine metabolism in Ames mice is markedly different than in their wild type littermates. In our previous work we hypothesized that the flux of methionine to the transsulfuration pathway is enhanced in the dwarf mice. The current study was designed to determine whether the flux of methionine to the transsulfuration pathway is increased. We did this by injecting either l-[methyl-(3)H]-methionine or l-[(35)S]-methionine into dwarf or normal mice and then determined retained label (in form of S-adenosylmethionine) 45 min later. The amount of retained hepatic (3)H and (35)S label was significantly reduced in the dwarf mice; at 45 min the specific radioactivity of SAM (pCi/nmol SAM) was 56% lower (p < 0.05) for (3)H-label and 64% lower (p < 0.005) for (35)S-label in dwarf than wild type mice. Retention of (35)S was significantly lower in the brain (37%, p < .04) and kidney (47%, p < 0.02) of the dwarf compared to wild type mice; there was no statistical difference in retained (3)H-label in either brain or kidney. This suggests that both the methyl-moiety and the carbon chain of methionine are lost much faster in the dwarf compared to the wild type mouse, implying that both transmethylation in the liver and transsulfuration in the liver, brain, and kidney are increased in the dwarf mice. As further support, we determined by real-time RT PCR the expression of methionine metabolism genes in livers of mice. Compared to wild type, the Ames dwarf had increased expression of methionine adenosyltransferase 1a (2.3-fold, p = 0.013), glycine N-methyltransferase (3.8-fold, p = 0.023), betaine homocysteine methyltransferase (5.5-fold, p = 0.0006), S-adenosylhomocysteine hydrolase (3.8-fold, p = 0.0005), and cystathionase (2.6-fold; tended to be increased, p = 0.055). Methionine synthase expression was significantly decreased in dwarf compared to wild type (0.48-fold, p = 0.023). These results confirm that the flux of methionine to transsulfuration is enhanced in the Ames dwarf. This, along with data from previous studies support the hypothesis that altered methionine metabolism plays a significant role in the oxidative defense of the dwarf mouse and that the mechanism for the enhanced oxidative defense may be through altered GSH metabolism as a result of the distinctive methionine metabolism.