Nephrocalcinosis (NC) in preterm neonates has been described frequently, and small-scale studies suggest an unfavorable effect on renal function. The etiologic factors have not yet been fully clarified. We performed a prospective observational study to identify factors that influence the development of NC. The study population consisted of 215 preterm neonates with a gestational age <32 weeks. Clinical characteristics and intake in the first four weeks of calcium, phosphorus, vitamin D, protein, and ascorbic acid were noted. Serum calcium, phosphate, vitamin D, magnesium, uric acid, creatinine, urea and urinary calcium, phosphate, oxalate, citrate, magnesium, uric acid, and creatinine were assessed at four weeks of age and at term. Renal ultrasonography (US) was performed at four weeks and at term. At term was defined as a postconceptional age of 38 to 42 weeks. NC was diagnosed by means of US in 33% at four weeks and in 41% at term. Patients with NC at four weeks had a significantly higher mean intake of calcium (P < 0.05), phosphorus (P < 0.05), and ascorbic acid (P < 0.01) than patients without NC. They had a higher mean serum calcium (2.55 vs. 2.46 mmol/L, P < 0.01) and a higher mean urinary calcium/creatinine ratio (2.6 vs. 2.1 mmol/mmol, P < 0.05). Patients with NC at term had a lower birth weight (1142 vs. 1260 g, P < 0.05) and a lower gestational age (28.8 vs. 29.4 weeks, P < 0.05), were treated significantly longer with furosemide, dexamethasone, theophylline, and thiazides, developed chronic lung disease more frequently (40 vs. 16%, P < 0.001), and had a higher mean urinary calcium/creatinine ratio (2.7 vs. 2.3 mmol/mmol, P < 0.05) and a lower mean urinary citrate/calcium ratio (1.1 vs. 1.7 mmol/mmol, P = 0.005). NC develops as a result of an imbalance between stone-inhibiting and stone-promoting factors. A high intake of calcium, phosphorus, and ascorbic acid, a low urinary citrate/calcium ratio, a high urinary calcium/creatinine ratio, immaturity, and medication to prevent or treat chronic lung disease with hypercalciuric side effects appear to contribute to the high incidence of NC in preterm neonates.