Coordinated expression patterns of genes controlling gluconeogenesis in primary hepatocytes suggested increased capacity for gluconeogenesis with increasing choline, but not methionine. The objective of this experiment was to quantify glucose export and cellular glycogen in primary bovine hepatocytes exposed to increasing concentrations of choline chloride (CC), d,l-Met (DLM), and added fatty acids (FA). Primary hepatocytes isolated from 3 Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (61, 128, 2028, 4528 μM) and DLM (16, 30, 100, 300 μM), with or without a 1 mM FA cocktail in a 4 × 4 × 2 factorial design. Treatments were applied in triplicate to basal medium with 5.5 mM glucose and 1.25 mM sodium pyruvate. After 24 h, medium was collected to quantify glucose, and cells were harvested to isolate glycogen and quantify DNA. Total glucose, comprised of medium glucose and glucose exported from cells, and total cellular glycogen were normalized to corresponding total DNA to account for differences in cell plating density between calves, before averaging the normalized values within triplicates. Data were expressed relative to the lowest CC and DLM treatment without FA within each cell prep before data were analyzed using PROC MIXED of SAS 9.4 with linear and quadratic contrasts in a model with fixed effect of treatment, corresponding interactions, and random effect of calf. Interactions were not significant; therefore, only main effects are discussed. Fatty acid treatment did not affect medium glucose (P = 0.98) or cellular glycogen (P = 0.29). Increasing CC linearly decreased (P = 0.0019) relative glucose in medium by up to 13.5%, and linearly (P > 0.0001) and quadratically (P < 0.0001) increased relative cellular glycogen by up to 26.7%. Medium glucose (P = 0.86) and cellular glycogen (P = 0.69) were not affected by DLM. Increased cellular glycogen with increasing CC, and a lack of change in medium glucose or cellular glycogen with increasing DLM, supports previous gene expression data. Differential effects of CC on cellular glucose export and cellular glycogen should continue to be investigated.