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To investigate the effects of age, sex and vitamin status on total plasma homocysteine (tHCy), both after fasting (FtHCy) and two hours post-methionine load (PML-tHCy). The secondary aim was to determine the reference values for FtHCy and PML-tHCy.A cohort of apparently healthy volunteers underwent blood sampling for FtHCy, PML-tHCy, creatinine, serum folate, vitamin B12 and vitamin B6 (pyridoxal-5-phosphate, PLP).In 147 subjects (M/F= 82/65, age range: 14-94 years), FtHCy was significantly higher in men than in women. In men, age and folate levels explained 20.5% and 19.0% of FtHCy variance, respectively. In women, age and vitamin B12 accounted for 22.6% and 17.8% of FtHCy variance, respectively. PML-tHCy was similar in men and women. PML-tHCy was negatively correlated with folate in both sexes, and with vitamin B12 and age in women only. Folate accounted for 20% of the variance of PML-tHCy in men, while in women vitamin B12 and PLP explained 40% and 20% of variance of PML-tHCy, respectively. The reference values of FtHCy and PML-tHCy were: 19.63 and 40.18 mol/L, respectively, for men under 45 years, 14.26 and 28.31 mol/L, respectively, for women under 45 years, 28.38 and 36.48 mol/L for men above 45 years, and 22.49 and 44.06 mol/L for women above 45 years.Age, gender and vitamin status influence both FtHCy and PML-tHCy in normal subjects. Reference values should be calculated according to age and sex.