Risk factors established at young ages may set the stage for later cardiovascular disease (CVD). Elevated total homocysteine (tHcy) in blood is an emerging risk factor for CVD, yet few studies have been conducted in children, especially in the Mediterranean. We described plasma tHcy concentrations in a group of healthy Greek children and examined its relation with physiologic, metabolic, and genetic variables. Fasting blood samples were collected from 186 students, 11.6 +/- 0.4 years old, and tHcy, folate, vitamin B-12, and routine biochemistry variables in plasma were measured. The methylenetetrahydrolate reductase (MTHFR) C677T genotype was determined and anthropometric and dietary data were obtained. The distribution of tHcy was positively skewed with a median of 7.9 micromol/L (mean: 8.2 +/- 2.3 micromol/L; range: 4.4-22.2 micromol/L). tHcy was inversely related to plasma folate (r = -0.34, P < 0.0001), vitamin B-12 (r = -0.20, P = 0.008), and glucose (r = -0.15, P = 0.045). An interaction between the MTHFR genotype and plasma folate on tHcy was detected (P = 0.047). Specifically, the homozygous mutant TT genotype was associated with higher tHcy only in children with lower plasma folate (< 19.9 nmol/L), (P = 0.012). In our sample of healthy Greek children, plasma tHcy concentrations were higher than values reported in children of Northern European descent and were associated with folate, vitamin B-12, and glucose in plasma. The results also show that, similar to adults, plasma folate concentration is important in determining the contribution of the MTHFR C677T mutation to tHcy concentrations in children.