Under-reporting of alcohol intake has been frequently reported. However, due to the lack of an objective reference method, e.g. a biomarker, information about the extent of under-reporting of alcohol intake obtained with dietary assessment instruments is not available. The objective of this study was to compare reported alcohol intake data derived from a 24 h recall with a biomarker of recent alcohol intake, the urinary excretion of 5-hydroxytryptophol (5-HTOL):5-hydroxyindole-3-acetic acid (5-HIAA). Embedded into the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study, Germany, a validation study that collected 24 h recall data and 24 h urine samples was conducted. Cohort study participants (n 107) volunteered to participate in this validation study. Among them were five subjects who reported no consumption of alcoholic beverages but had a 5-HTOL:5-HIAA ratio that indicated recent alcohol intake when the clinical cut-off point was taken as a judging criterion. After exclusion of these under-reporters, the Pearson's correlation coefficient between reported alcohol intake and the 5-HTOL:5-HIAA ratio was 0.92 (P<0.0001). Except for low alcohol intake of <0.1 g/kg body mass, a significant increase in 5-HTOL:5-HIAA excretion was observed with increasing amounts of alcohol intake. In conclusion, the 5-HTOL:5-HIAA excretion ratio appears to be a valuable quantitative biomarker of recent alcohol consumption. Denial of alcohol intake can be detected, but for the quantification of under-reporting of alcohol intake 24 h reference data are not yet available. With these data at hand, however, 5-HTOL:5-HIAA could become a biomarker for validation purposes in nutritional epidemiology.