Homocysteine is a precursor of S-adenosylmethionine (AdoMet) and a metabolite of S-adenosylhomocysteine (AdoHcy). The ratio of AdoMet / AdoHcy, defined as the methylation potential (MP), indicates the flow of methyl groups within the cells. Chronic elevations of total homocysteine (tHcy) in plasma correlate with increased AdoHcy concentrations, decreased MP, and impaired DNA methylation. However, the influence of acute hyperhomocysteinemia on MP is unknown. We induced acute hyperhomocysteinemia in 14 healthy volunteers by oral administration of L-homocysteine (65.1 µmol/kg of body weight) in an open, randomized, placebo-controlled 2-period cross-over study. The kinetics of tHcy in blood and urine, MP in blood, and global DNA methylation in lymphocytes were studied systematically during 48 hours. Plasma tHcy concentrations reached a peak at 34 ± 11 min (mean ± SD) after oral load with L-homocysteine and decreased with a half-life of 257 ± 41 min (mean ± SD). Only 2.3% of the homocysteine dose were recovered in urine. AdoHcy concentrations and MP in whole blood and erythrocytes were not affected by oral homocysteine load. Furthermore, global DNA methylation in lymphocytes did not change under these conditions. We found no difference between the genotypes of 5,10-methylenetetrahydrofolate reductase (MTHFR) in response to the homocysteine load. However, AdoMet content in erythrocytes was significantly higher in the C677T carriers (CT; n=7) compared to the CC genotype (n=7). Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of plasma tHcy above 20 µmol/l for 8 hours is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes.