Nutrient effects on cholesterol fractional synthesis rates (FSR) in infancy by stable isotope determination have not been studied. We hypothesized that FSR is significantly reduced with high dietary cholesterol and phytoestrogen intake and increased with low dietary cholesterol and phytoestrogen intake. We prospectively studied 33 term male infants exclusively fed human milk (high cholesterol, low phytoestrogen, n = 12), cow milk-based formula (low cholesterol, low phytoestrogen, n = 8), soy milk-based formula (zero cholesterol, high phytoestrogen, n = 7), or soy milk-based formula modified to contain cholesterol (low cholesterol, high phytoestrogen, n = 6) during the first 4 mo of life. Cholesterol FSR was determined from rate of incorporation of deuterium into erythrocyte membrane cholesterol, and urinary isoflavone excretion (an index of dietary phytoestrogen exposure) was measured by gas chromatography-mass spectrometry. Significant differences in cholesterol FSR were found. FSR (%/d) was lowest in human milk (2.62 ± 0.38), highest in soy milk-based formula (9.40 ± 0.51), and intermediate in cow milk-based and modified soy milk-based formula (6.90 ± 0.48 and 8.03 ± 0.28, respectively), p < 0.0001. Cholesterol FSR was significantly lower in modified soy milk-based compared with soy milk-based formula, p < 0.05. We also show for the first time that dietary phytoestrogens are absorbed and excreted by the infant fed soy protein-based formula. Urinary isoflavone excretion was inversely related to cholesterol FSR, but it was not significantly related to serum cholesterol concentration. We conclude that the type of infant nutrition and dietary cholesterol are major factors influencing cholesterol fractional synthesis rates in infancy.