Background: A high plasma concentration of total homocysteine (tHcy) is associated with increased risk of cardiovascular disease. A high protein intake and hence a high intake of methionine-the sole dietary precursor of homocysteine-may raise plasma tHcy concentrations. Objectives: We studied whether high intake of protein increases plasma concentrations of tHcy in the fasting state and throughout the day. Design: We conducted a randomized, dietary controlled, crossover trial in 20 healthy men aged 18-44 y. For 8 d, men consumed a controlled low-protein diet enriched with either a protein supplement [high-protein diet (21% of energy as protein)] or an isocaloric amount of short-chain glucose polymers [low-protein diet (9% of energy as protein)]. After a 13-d washout period, treatments were reversed. On days 1 and 8 of each treatment period, blood was sampled before breakfast (fasting) and throughout the day. Results: Fasting tHcy concentrations did not differ significantly after the 1-wk high-protein and the 1-wk low-protein diets. The high-protein diet resulted in a significantly higher area under the 24-h homocysteine-by-time curves compared with the low-protein diet, both on day 1 (difference: 45.1 h · μmol/L; 95% CI: 35.3, 54.8 h · μmol/L; P < 0.0001) and on day 8 (difference: 24.7 h · μmol/L; 95% CI: 15.0, 34.5 h · μmol/L; P < 0.0001). Conclusions: A high-protein diet increases tHcy concentrations throughout the day but does not increase fasting tHcy concentrations. As previously shown, the extent of the tHcy increase is modified by the amino acid composition of the protein diet. The clinical relevance of this finding depends on whether high concentrations of tHcy-particularly postprandially-cause cardiovascular disease. © 2005 American Society for Clinical Nutrition.