A common mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene results in elevated homocysteine levels and, presumably, in increased atherosclerotic risk. We evaluated serum homocysteine levels, MTHFR genotype, and a panel of variables in a sample of 155 middle-aged Italian subjects (mean age 38.1 years). Biometrical, hematological, and biochemical variables (including serum folate and vitamin B12) and lifestyle characteristics were investigated. MTHFR genotype was studied by polymerase chain reaction. The frequency of the genotype Val/Val (homozygosity for the mutant allele) was 16.13%. The Val/Val genotype was associated with increased levels of homocysteine; no differences among genotypes were seen in individuals with folate or vitamin B12 levels at or above the median values. In multivariate analysis, MTHFR genotype was an independent predictor of homocysteine levels in both biochemical and non biochemical regression models. Sex and diastolic blood pressure emerged as non biochemical variables independently associated with homocysteine. Apart from cofactors, uric acid was the only biochemical variable independently associated with homocysteine, particularly in subjects with Val/Val genotype. The observed parallel increases in homocysteine and uric acid levels in subjects with thermolabile MTHFR warrant further investigation.