BHMT transfers a methyl group from betaine (Bet) to homocysteine to form Met and dimethylglycine. Bet is obtained from the diet or by the endogenous oxidation of choline by CHDH. BHMT is expressed in rat liver and is regulated by dietary Met and choline. BHMT is also found in rat kidney, albeit in substantially lower amounts, but it is not known whether it is regulated by dietary Met or choline. Whether diet has a role in the regulation of CHDH expression has not been investigated. Rats (~50g) were fed (9 days) in 3 x 2 factorial design (n = 8) a purified diet varying in Met (0.125, 0.3 or 0.8%) and choline (0 or 0.625%). Liver and kidney BHMT and CHCH expression were assessed using enzymatic, Western blot and real-time PCR analyses. Livers were also fixed in 10% buffered formalin for histological analysis. Animals fed low Met diets had significantly lower feed efficiencies. A degree of fatty liver was observed in all rats fed a choline-devoid diet, indicating that excess Met cannot compensate for low dietary choline. Compared to rats fed the 0.3% Met and 0.625% choline diet, liver BHMT activity was 113% higher in animals fed the 0.125% Met containing 0.625% choline and was also reflected in increases in mRNA content (2.7-fold) and immunodetectable protein. Excess dietary Met with or without choline increased liver BHMT activity 19.7% to 33.3%. Kidney BHMT was refractory to dietary treatment. Liver CHDH immunodetectable protein and mRNA content were not affected by dietary Met and choline availability. This work was supported by NIDDK (DK52501).