Overlap in hepatic methyl pathways highlights a role for potential competition and compensation of methyl donors. The objective of this experiment was to examine the regulation of genes controlling methyl group transfer in response to increasing concentrations of choline chloride (CC), dl-Met (dLM), and added fatty acids (FA). Primary hepatocytes isolated from 4 Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (33, 100, 200, 450 μM) and dLM (16, 30, 100, 300 μM), with or without a 1 mM FA cocktail in a factorial design. Concentrations mimicked expected physiological concentrations. After 24 h, media was collected for quantification of reactive oxygen species (ROS) by fluorometric assay and cells were collected for quantification of gene expression. Data were analyzed using PROC MIXED of SAS 9.4 with linear and quadratic contrasts in a model with fixed effect of treatment and random effects of calf. Interactions were not significant and therefore only main effects are discussed. Met can be generated from betaine via BHMT, or from homocysteine via MTR which also serves as the final step in regeneration of met after methyl-group donation. Increasing dLM concentration did not alter BHMT expression but did decrease (P = 0.003) MTR expression. Increasing concentration of CC did not alter BHMT but did increase (P = 0.02) MTR expression, suggesting that CC plays a key role in regeneration of met after methyl group donation. FA increased (P = 0.05) BHMT expression but decreased (P = 0.0001) MTR expression, which favors regeneration of met that is coupled with downstream glutathione production, which may aid the cell with oxidative stress associated with FA metabolism. Both CC and dLM decreased (P = 0.02) expression of MAT1A, the enzyme that generates SAM from met. PEMT expression was not affected by CC or dLM suggesting that dLM was not used to generate phosphatidylcholine. ROS tended to decrease (P = 0.08) with increasing CC treatment but was not changed with dLM treatment. These data suggest that CC may play a critical role in donating methyl groups and in decreasing ROS within the hepatocyte.