Choline and methionine can both serve as methyl-donors; however, previous works suggests they may serve unique functions to regulate carbon metabolism within bovine hepatocytes. The objective of this experiment was to trace the flux of carbon through pathways of fatty acid (FA) metabolism in bovine hepatocytes exposed to choline and methionine. Primary hepatocytes isolated from 3 neonatal Holstein calves were maintained as monolayer cultures for 24 h. At 24 h, media was refreshed and treatments of choline chloride (CC; 0, 0.01, 0.1, or 1.0 mM) and d,l-Met (DLM; 0, 0.1, or 0.3 mM) were applied in a factorial design along with 1.0 mM FA that reflected the blood FA profile at calving. After 21 h of treatment, [1-¹⁴C]palmitate was added to the media and both CO₂ and acid soluble products (ASP) were collected after a 3h incubation. Parallel treatments were incubated without radiolabeled substrate for 24h to quantify media reactive oxygen species (ROS) and cellular triglyceride (TG). Cellular oxidation and TG were normalized to DNA. Data were expressed relative to a no FA/CC/DLM control within each cell prep, and analyzed by PROC MIXED (SAS 9.4) in a model accounting for fixed effects of CC, DLM, interaction of CC and DLM, and random effect of calf. Contrasts evaluated for CC were 0 vs. (0.01, 0.1, and 1.0 mM) and linear contrast 0.01 vs. 0.1 vs. 1.0 mM. Contrasts evaluated for DLM were: 0 vs. (0.1 and 0.3 mM) and 0.1 vs. 0.3 mM. Data are reported as least squares means ± SE and differences declared at P ≤ 0.10 and tendencies at P < 0.15. No interactions of CC and DLM were detected. DLM did not alter (P > 0.15) cellular TG, ROS, or recovery of substrate label as CO₂ or ASP. Recovery of palmitate label as CO₂ or ASP was not altered (P > 0.15) by CC treatment, indicating no change in complete or incomplete oxidation of FA with CC treatment. Presence of CC decreased (P = 0.03) cellular TG accumulation (3.3 vs. 2.9 ± 0.03 arbitrary units) and concomitant secretion of ROS into media (P = 0.04). Treatment with CC did not affect fatty acid oxidation but exhibited a hepatoprotective effect by decreasing cellular TG accumulation and ROS secretion, despite methionine treatment.