Background: Homocysteine (Hcy) levels are modulated by nutritional and genetic factors, among which is the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Aim: To determine the effects of the MTHTR C677T polymorphism, as well as the intake of folate, vitamins B-6 and B-12 on serum Hcy concentration in the Polish population. Methods: Within the framework of the National Multicentre Health Survey (WOBASZ), a representative sample of the whole Polish population aged 20-74 was screened in 2003-2005. Vitamins intake, Hcy level and known MTHTR C677T genotype were available for 1,561 men and 1,712 women. Results: In the Polish population, T/T, C/T and C/C genotype frequencies were 10%, 43% and 47%, respectively in men, and 9%, 42% and 49%, respectively in women. The T/T genotype was associated with increased levels of Hcy (13.14 mu mol/L in men, and 9.77 mu mol/L in women) compared to the C/C and C/T genotypes (10.18 and 8.77, respectively), after adjustment for age, methionine, coffee and alcohol intake, smoking and drugs used. In a multivariable linear regression model, among subjects with the T/T genotype, the only factor influencing Hcy was age in women. In the case of the other groups (C/C and C/T), there was a relationship between Hcy and age, alcohol consumption, drugs used, folate and vitamin B-6 in men, and age, smoking, coffee consumption, drugs used, folate and vitamin B-12 in women. Conclusions: The T/T genotype is associated with higher levels of Hcy (29% in men, and 11% in women) compared to other genotypes. Nutritional factors affect Hcy levels only in the C/C and C/T MTHFR genotypes.