Background: Choline is essential for mammalian cell function. It plays a critical role in cell membrane integrity, neurotransmission, cell signaling and lipid metabolism. Moreover, choline is involved in methylation in two ways: a) its synthesis requires methyl groups donated by S-adenosyl-methionine (AdoMet); and b) choline oxidation product betaine methylates homocysteine (Hcy) to methionine (Met) and produces dimethylglycine. This later donates one carbon units to tetrahydrofolate (THF). Methods: To evaluate the correlations of choline and betaine with folate, AdoMet, S-anenosyl-homocysteine (AdoHcy), total homocysteine (tHcy), and DNA methylation, choline, betaine and dimethylglycine were measured by LC-MS/MS in plasma of 109 healthy volunteers, in whom folate, AdoMet, AdoHcy, tHcy, and DNA methylation have previously been reported. Results: Using a bivariate model, choline and betaine showed strong positive correlations with folate (r=0.346 and r=0.226), AdoHcy (r=0.468 and r=0.296), and correlated negatively with AdoMet/AdoHcy ratio (r=–0.246 and r=–0.379). Only choline was positively correlated with AdoMet (r=0.453). Using a multivariate linear regression model, choline correlated strongly with folate (β=17.416), AdoMet (β=61.272), and AdoHcy (β=9.215). Betaine correlated positively with folate (β=0.133) and negatively with tHcy (β=–0.194) ratio. Choline is an integral part of folate and methylation pathways. Conclusions: Our data highlight the importance of integrating choline in studies concerning addressing pathological conditions related to folate, homocysteine and methylation metabolism.