Experiments were conducted to assess the significance of de novo choline synthesis in the oxidative metabolism of the methyl group of L-methionine in the rat. Comparison of the rates of 14CO2 production by rats fed diets containing equimolar or equal methyl loads of [methyl-14C]methionine, [methyl-14C] choline, [methyl-14C] betaine and [methyl-14C] sarcosine revealed that the methyl groups of choline, betaine and sarcosine were oxidized at rates nearly equivalent to or exceeding that of the methionine methyl group. However, the results of a second series of in vivo experiments using non-radioactive choline or betaine as intermediary trapping pools indicated that little newly formed radioactive choline was released from tissue phospholipid choline during the period of rapid 14CO2 production from [14CH3] methionine. Incorporation of the methionine methyl group into phospholipid choline by incubated liver slices was strongly inhibited by 2-methoxyethanol without a parallel inhibition of methionine methyl group oxidation. Results of these in vitro studies also indicated that the metabolic pathway responsible for the transfer of the methionine methyl group into choline and that responsible for its oxidation to CO2 differ widely in the ease with which they can be saturated by L-methionine. It is concluded that essentially all of the methionine methyl group oxidation in vitro and that occurring during the absorptive period in vivo must depend primarily on pathways other than those leading to the synthesis of choline.