To test the hypothesis that estrogen modulates the metabolism of 25-hydroxyvitamin D (25(OH)D) to 1,25-dihydroxyvitamin D (1,25(OH)2D) and 24,25-dihydroxyvitamin D (24,25(OH)2D), we studied 20 normal premenopausal women at four consecutive weekly intervals during one menstrual cycle. Estrogen stimulation was semiquantitatively defined into baseline, low-grade, or medium-grade categories, based on endogenous estrone and estradiol concentrations. 1,25(OH)2D increased incrementally from baseline levels of 34 +/- 3(SE) pg/ml to 39 +/- 3 pg/ml (P = 0.2) with low-grade estrogen stimulation and to 43 +/- 3 pg/ml (P less than 0.05) with medium-grade estrogen stimulation, while 25(OH)D, 24,25(OH)2D, vitamin D binding protein, parathyroid hormone, calcium, and phosphate did not change. 24,25(OH)2D was correlated to 25(OH)D at baseline (r = 0.65, P less than 0.01) and with low-grade estrogen stimulation (r = 0.62, P less than 0.01), but not with medium-grade stimulation (r = 0.13); these relationships are consistent with the concepts that 25(OH)D is metabolized predominantly to 24,25(OH)2D at low estrogen levels, but not at higher estrogen levels. We conclude that endogenous estrogen elevation promotes formation of 1,25(OH)2D from 25(OH)D, and that it may reciprocally inhibit synthesis of 24,25(OH)2D.