We performed a cross-sectional study (n = 49 males, 57 females) and a randomized double-blind placebo-controlled dietary intervention study (n = 31/32 per group) to determine the effect of folate and vitamin B12 (B12) on DNA damage (micronucleus formation and DNA methylation) and plasma homocysteine (HC) in young Australian adults aged 18-32 years. None of the volunteers were folate deficient (i.e. red blood cell folate <136 nmol/l) and only 4.4% (all females) were vitamin B12 deficient (i.e. serum vitamin B12 <150 pmol/l). The cross-sectional study showed that (i) the frequency of micronucleated cells (MNCs) was positively correlated with plasma HC in males (R = 0.293, P < 0.05) and (ii) in females MNC frequency was negatively correlated with serum vitamin B12 (R = -0.359, P < 0.01) but (iii) there was no significant correlation between micronucleus index and folate status. The results also showed that the level of unmethylated CpG (DNA) was not significantly related to vitamin B12 or folate status. The dietary intervention involved supplementation with 3.5x the recommended dietary intake (RDI) of folate and vitamin B12 in wheat bran cereal for three months followed by ten times the RDI of these vitamins via tablets for a further three months. In the supplemented group, MNC frequency was significantly reduced during the intervention by 25.4% in those subjects with initial MNC frequency in the high 50th percentile but there was no change in those subjects in the low 50th percentile for initial MNC frequency. The reduction in MNC frequency was significantly correlated with serum vitamin B12 (R = -0.49, P < 0.0005) and plasma HC (R = 0.39, P < 0.006), but was not significantly related to red blood cell folate. DNA methylation status was not altered in the supplemented group. The greatest decrease in plasma HC (by 37%) during the intervention was observed in those subjects in the supplemented group with initial plasma HC in the high 50th percentile, and correlated significantly with increases in red blood cell folate (R = -0.64, P < 0.0001) but not with serum vitamin B12. The results from this study suggest that (i) MNC frequency is minimized when plasma HC is below 7.5 micromol/l and serum vitamin B12 is above 300 pmol/l and (ii) dietary supplement intake of 700 microg folic acid and 7 microg vitamin B12 is sufficient to minimize MNC frequency and plasma HC. Thus, it appears that elevated plasma HC, a risk factor for cardiovascular disease, may also be a risk factor for chromosome damage.