Preterm infant formulas (PIFs) for very-low-birth-weight (VLBW) infants (birth weight, < 1,500 g) are augmented to provide daily riboflavin and pyridoxine at levels up to five-fold greater than in term infant formula and 18-fold greater than in human milk. We evaluated plasma riboflavin and pyridoxine concentrations in VLBW infants who received PIF during their first postnatal month. Eighty-eight plasma and 124 urine samples were collected for riboflavin- and pyridoxine-concentration measurements from 57 clinically healthy VLBW infants weekly during their first postnatal month. Concentrations were measured using high-performance liquid chromatography. At the time of the sample, patients were receiving > or = 80% of their total calories via enteral feedings. Plasma riboflavin concentrations rose from 45.3 +/- 7.3 ng/ml at baseline (mean +/- SEM) to 173.5 +/- 20.3 ng/ml by 1 week of age and remained at 177.3-199.7 ng/ml during the following three weekly measurements; values were up to 14-fold above baseline concentration. Urine riboflavin concentration increased from 534 +/- 137 ng/ml at baseline to 3,521 +/- 423 ng/ml by 1 week of age and remained at 4,451-5,216 ng/ml during the next 3 weeks. In a similar pattern, baseline plasma (69.4 +/- 10.4 ng/ml) and urine (145 +/- 30 ng/ml) pyridoxine concentrations were significantly increased by 1 week postnatal age; they remained at 163-248 ng/ml (plasma) and 1,573-2,394 ng/ml (urine) through the first postnatal month. Plasma and urine riboflavin and pyridoxine concentrations in enterally fed VLBW infants increased from baseline concentrations by 1 week of postnatal age and remained elevated for the first postnatal month. High daily intake and immature renal development are probable contributing causes of the elevated plasma riboflavin and pyridoxine concentrations. We suggest that lower daily enteral administration of riboflavin and pyridoxine should maintain adequate blood concentrations and minimize potential toxicity.