Little is known about the bioavailability of isoflavones in children. Previous studies have shown that children excrete more isoflavone in urine compared with adults. Thus we examined the relationship between usual dietary isoflavone intake and the urinary excretion of isoflavonoids in Korean girls of pubertal age. Twelve girls each were selected from the lowest and the highest quartiles of isoflavone intake among 252 Korean girls aged 8–11 years. Age, BMI and sexual maturation stage were matched between the two groups. Dietary intakes for 3 d by diet record and overnight urine samples were collected at baseline and at 6 and 12 months. Total and individual isoflavone (daidzein, genistein and glycitein) intakes were calculated from diet records. The parent isoflavone compounds (daidzein, genistein and glycitein) and their metabolites (equol, O-desmethylangolensin (O-DMA), dihydrodaidzein and dihydrogenistein) present in the urine samples were analysed using liquid chromatography–MS. Intake levels of total and individual isoflavone compounds were significantly higher in the high isoflavone (HI) group than the levels in the low isoflavone (LI) group (P < 0·05). Urinary excretion of all isoflavone parent compounds was significantly higher in the HI group than in the LI group (P < 0·0001). Among isoflavone metabolites, only O-DMA and total metabolites were significantly different (P < 0·05). Total isoflavone intake was highly correlated with the urinary excretion of total parent compounds (r 0·68; P < 0·01), parent compounds plus their metabolites (r 0·66–0·69; P < 0·01) and total isoflavonoids (r 0·72; P < 0·0001). In conclusion, overnight urinary excretion of total isoflavonoids is a reliable biomarker of usual isoflavone intake in Korean girls of pubertal age.