Summary Little information is available to estimate water-soluble vitamin intakes from urinary vitamins and their metabolite contents as possible nutritional markers. Determination of the relationships between the oral dose and urinary excretion of water-soluble vitamins in human subjects contributes to finding valid nutrition markers of water-soluble vitamin intakes. Six female Japanese college students were given a standard Japanese diet in the first week, the same diet with a synthesized water-soluble vitamin mixture as a diet with approximately onefold vitamin mixture based on Dietary Reference Intakes (DRIs) for Japanese in the second week, with a threefold vitamin mixture in the third week, and a sixfold mixture in the fourth week. Water-soluble vitamins and their metabolites were measured in the 24-h urine collected each week. All urinary vitamins and their metabolite levels except vitamin B 12 increased linearly in a dose-dependent manner, and highly correlated with vitamin intake ( r ϭ 0.959 for vitamin B 1 , r ϭ 0.927 for vitamin B 2 , r ϭ 0.965 for vitamin B 6 , r ϭ 0.957 for niacin, r ϭ 0.934 for pantothenic acid, r ϭ 0.907 for folic acid, r ϭ 0.962 for biotin, and r ϭ 0.952 for vitamin C). These results suggest that measuring urinary water-soluble vitamins and their metabolite levels can be used as good nutritional markers for assessing vitamin intakes. Key Words biomarker, human, urine, vitamin A nutritional marker can be an indicator of nutritional status with respect to intake or metabolism of dietary constituents. Nutritional markers can be designated into one or more of three categories, 1) a means of validation of dietary instruments, 2) surrogate indicators of dietary intakes, or 3) integrated measures of nutritional status for a nutrient ( 1 ). Nutritional markers may be interpreted more broadly as a biological consequence of dietary intake or dietary patterns, and contribute to setting recommendations, tolerable levels and guidelines. Recent validation studies have developed the urinary compounds as nutritional markers to estimate nutrient intakes. For example, 24-h urinary nitrogen has been established as a marker for protein intake ( 2 ), the same as urinary potassium for energy and potassium intake ( 3 ), and urinary sugars for sugar intake ( 4 ).Water-soluble vitamins are absorbed from the digestive tract after ingestion, stored in the liver, delivered to peripheral sites and then excreted to urine. Urinary water-soluble vitamins or their metabolites decrease markedly as vitamin status declines, and they are affected by recent dietary intake. Urinary excretion of water-soluble vitamins such as thiamin, riboflavin and niacin has been used for setting Dietary Reference Intakes (DRIs) in the USA and Japan ( 5 , 6 ). However, only a single study investigated urinary vitamins as a possible marker for intake. Individuals' 30-d means of thiamin intake are highly correlated with their mean 24-h urine thiamin levels under strictly controlled conditions, showing 24-h urinary thiamin as a useful m...