Guanidinoacetic acid (GAA) is the precursor of creatine, which is produced by hepatic methylation of GAA. We evaluated the metabolic response of cattle to post-ruminal supplementation of GAA with and without methionine (Met) supplementation as a source of methyl groups. Six ruminally cannulated Holstein heifers (520 ± 49 kg initial BW) were fed twice daily a diet containing 6 kg/d rolled corn, 4 kg/d alfalfa hay, and 50 g/d trace-mineralized salt. The experiment used a split-plot design. All treatments were infused continuously to the abomasum. The main plot treatments were 0 or 12 g/d of L-Met arranged in a completely randomized design; 3 heifers received each treatment throughout the entire experiment. Subplot treatments were 0, 10, 20, 30, and 40 g/d of GAA, with treatments provided in sequence from lowest to highest level using 6-d periods for each level. Blood and urine samples were collected on d 6 of each period. Met supplementation increased plasma Met (P < 0.01). GAA supplementation linearly increased plasma Arg (% of total AA), suggesting a sparing of Arg for GAA synthesis. GAA supplementation linearly increased plasma concentrations of GAA and creatinine (P < 0.001) and increased plasma creatine at all levels of GAA except when 40 g/d of GAA was supplemented with no Met (GAA quadratic × Met, P = 0.07). Plasma homocysteine was not affected by GAA supplementation when heifers received 12 g/d Met, but it was increased in response to 30 or 40 g/d of GAA when no Met was supplemented (GAA linear × Met, P = 0.003); increases did not suggest a dangerous hyperhomocysteinemia. Urinary GAA and creatine concentrations were increased by all levels of GAA when 12 g/d Met was provided and by GAA supplementation up to 30 g/d when no Met was provided, but 40 g/d GAA did not increase urinary concentrations of GAA or creatine when no Met was supplemented (GAA quadratic x Met, P ≤ 0.06). Data suggest that post-ruminal GAA supplementation increased creatine supply to cattle. An apparent methyl group deficiency, demonstrated by increases in plasma homocysteine, developed when 30 or 40 g/d of GAA was supplemented, but it was ameliorated by 12 g/d Met.