Malondialdehyde (MDA) is one of the most frequently used indicators of lipid peroxidation. To generate reliable reference intervals for plasma malondialdehyde (P-MDA), a reference sample group was established in Funen, Denmark. The group consisted of 213 individuals (107 men, 106 women), ages 20-79 years. P-MDA was measured in EDTA-treated plasma after derivatization by thiobarbituric acid (TBA) and separation on HPLC. UV detection was performed at 532 nm. A reference interval was calculated as recommended by IFCC with REFVAL 3.42. The estimated reference limits (0.025 and 0.975 fractals) for the group were 0.36 and 1.24 mumol/L. The data were analyzed for gender- and age-related differences. Analysis of variance showed no interaction between gender and age, but separate analyses showed an independent effect of gender (P = 0.03), but not of age (P = 0.11). Daily smokers had a slightly higher average concentration of P-MDA than nonsmokers (P = 0.05), and P-MDA correlated with daily exposure to cigarette smoke (r = 0.162; P = 0.03). A positive correlation was also demonstrated between P-MDA and weekly alcohol consumption (r = 0.153; P = 0.03). Within-subject and day-to-day variations of P-MDA indicated that the potential of P-MDA as a biomarker for individuals is questionable. However, on a group basis, the present data support that P-MDA may be a potential biomarker for oxidative stress.