Hyperhomocysteinemia is a risk factor for cardiovascular and other diseases. Recently many endogenous and exogenous modulators of homocysteine (Hcy) have become known, e.g., B-vitamins. However, little is known about the effect of exercise on Hcy. The purpose of this study was to investigate the effect of three different types of acute endurance exercise on serum Hcy. We measured Hcy in 100 recreational athletes (87 males, 13 females) who participated in a marathon race (n = 46), a 100 km run (100 km; n = 12) or a 120 km mountain bike race (n = 42). Blood samples were taken before, 15 min and 3 h after the race. In athletes with pre-race Hcy > 12 micromol/l we also determined folate and vitamin B12. Marathon running induced a Hcy increase of 64%, while mountain biking and 100 km running had no significant effect on Hcy. Pre-race Hcy (25th-75th percentile) overall; marathon race; 100 km; mountain bike race was 9.7 (7.1-11.5) micromol/l; 9.8 (7.4-11.1) micromol/l; 10.2 (6.6-13.2) micromol/l; 9.1 (6.9-13.5) micromol/l, respectively. At 15 min and 3 h post-race, Hcy was 11.9 (8.4-16.4) micromol/l; 16.1 (12.7-20.4) micromol/l; 9.5 (7.8-15.9) micromol/l; 8.8 (7.1-11.2) micromol/l, respectively, and 11.5 (8.9-15.7) micromol/l; 14.9 (11.5-20.0) micromol/l; 10.0 (8.1-11.8) micromol/l; 9.4 (7.4-12.1) micromol/l, respectively. The change in Hcy correlated negatively with the running time. Twenty-three athletes had pre-race Hcy levels > 12 micromol/l, which were associated with relatively low folate (14.3 (11.6-18.9) nmol/l) and vitamin B12 levels (231 (183-261) pmol/l). Endurance exercise may induce a considerable Hcy increase, which varies between different disciplines and is most probably determined by the duration and intensity of exercise. Furthermore, about 25% of recreational endurance athletes exhibited hyperhomocysteinemia in association with low vitamin B12 and folate levels.