To assess the variability of plasma homocysteine levels, fasting and post-methionine homocysteine levels were measured twice, at baseline and after follow-up of 1-4 months, in 16 individuals with normal and 26 with elevated homocysteine levels after methionine loading. The intra-individual coefficients of variation varied from 15 to 23% for fasting and post-methionine homocysteine levels, whether these levels were within the normal range or not. As a result, test-retest agreement was poor when subjects were dichotomized as having 'normal' or 'abnormal' homocysteine levels (itself a questionable concept). There was a relation between the average post-methionine homocysteine levels (at the first and second measurement) and the difference between both measurements (r = 0.37, P = 0.016). In normohomocysteinaemic individuals, delta (i.e., the difference between baseline and follow-up) fasting homocysteine and delta post-methionine homocysteine were correlated negatively with delta folate serum levels: r = -0.64, P = 0.007 and p = -0.50, P = 0.05, respectively. Individuals homozygous for the 677 C→T mutation in the 5,10-methylenetetrahydrofolate reductase gene showed a greater variation of fasting homocysteine levels than those homozygous for the wild type (P = 0.017). In summary, we suggest that there is a substantial intra-individual variability in plasma homocysteine levels over time and that this variability is significantly related to the variability in serum folate levels, especially in normohomocysteinaemic individuals.