The effects of chronic betaine ingestion were investigated in rats. Rats were fed an experimental diet containing 5% betaine for 4 wk and methionine was intravenously administered. The elevations of plasma homocysteine were assessed by comparing the increments to the initial measured value and the positive incremental area under the plasma homocysteine concentration curve over the 240-min post-methionine-loading period (deltaAUC0-240). In the betaine-ingesting rats, deltaAUC0-240 was significantly lower than in the control group (48% of the control), and the increments of plasma homocysteine were also lower compared with the control, especially 15-30 min after methionine loading. Choline, a precursor of betaine, did not alter the plasma homocysteine elevation. In a definite period immediately after methionine loading, carnitine, a methyl-group-rich amino acid, induced a significant increase of plasma homocysteine, compared to the control. Moreover, plasma homocysteine concentration was significantly decreased by 4 wk of betaine ingestion. Betaine enhanced liver BHMT activity whereas choline and carnitine did not show any effects on BHMT activity. These results suggest that betaine contributes to both the decrease in the plasma homocysteine concentration and the suppression of plasma homocysteine elevation through the activation of liver BHMT.