The response of osteocalcin and other biochemical markers of vitamin K status to diets formulated to contain different amounts of phylloquinone was assessed in nine healthy subjects aged 20-33 y. Subjects resided in a metabolic ward for two 15-d cycles with a minimum of 6 wk between cycles. A mixed diet containing 100 micrograms phylloquinone/d was fed throughout both cycles; however, the phylloquinone content of one of the cycles was increased to a total of 420 micrograms/d on days 6 through 10 by fortifying corn oil in the diet with phylloquinone (supplemented diet). Total serum osteocalcin concentrations were not affected by either of the dietary treatments. The percentage of undercarboxylated osteocalcin increased an average of 28% over the 15-d cycle with the mixed diet (P < 0.05) and declined significantly an average of 41% with 5 d of the supplemented diet (day 6: 21.9 +/- 1.3%, day 11: 12.8 +/- 1.1%; P = 0.0001) with a rise after the return to the mixed diet (16.7 +/- 1.3%, P < 0.001). Plasma phylloquinone concentrations increased significantly with supplementation (day 6: 0.95 +/- 0.16 nmol/L, day 11: 1.40 +/- 0.29 nmol/L; P < 0.001) and then rapidly returned to presupplementation concentrations on returning to the mixed diet. Twenty-four-hour ratios of urinary gamma-carboxyglutamic acid to creatinine were unchanged with the supplemented diet; however, excretion declined to 91 +/- 2% of baseline after 10 d on the mixed diet (P = 0.01). These results show that undercarboxylated osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid excretion appear to be sensitive measures of vitamin K nutritional status because all of these variables were responsive to changes in dietary intake.