Amphotericin B (AmphoB) remains the preferred therapy for invasive fungal infections despite many side effects, such as nephrotoxicity and electrolyte imbalance. Our previous study suggested that high sodium (Na) intake >4 mEq/kg per day may be associated with lower nephrotoxicity in extremely premature infants treated with AmphoB. Subsequently, it became a standard of care in our unit to administer Na >4 mEq/kg per day to extremely premature infants treated with AmphoB. The purpose of this study was to evaluate the effect of high Na intake > 4 mEq/kg per day on the incidence of AmphoB-induced nephrotoxicity among extremely premature infants with birth weight <1250 gm. All extremely premature infants with birth weight <1250 gm born between 1992 and 2004 and treated with AmphoB for systemic fungal infections were included in the study. The study infants were divided into two groups: a control (CL) group (1/1992-12/1999, n = 21) consisting of extremely premature infants given a maintenance Na intake during AmphoB therapy, and a high sodium intake (High Na) group (1/2000-12/2004, n = 16) consisting of extremely premature infants given a high Na intake >4 mEq/kg per day during AmphoB therapy. Nephrotoxicity was defined as serum creatinine levels >1 mg/dl, urinary output (UOP) < 1 ml/kg per hour or a decrease in UOP of 50%, compared with the previous 2 days, and persisting for at least 2 days. Invasive fungal infection was diagnosed in 5.7% of the infants (44/763 infants). Thirty-seven infants were eligible for the study and seven were excluded. There were no differences between the two groups in gestational age, birth weight, age at fungal infection diagnosis, length of AmphoB therapy, daily fluid intake or hyponatremia. Nephrotoxicity was significantly higher in the CL group than in the High Na group (13/21 vs. 3/16; P = 0.02). In the CL group, nephrotoxicity occurred at (mean +/- SD) 1.9 +/- 3.2 days after the initiation of AmphoB treatment and lasted for 5.5 +/- 4.7 days. In this group, nephrotoxicity occurred in two of the 13 infants before the initiation of AmphoB therapy. In the High Na group, nephrotoxicity occurred before the start of AmphoB therapy in two of the three infants. In the third infant, nephrotoxicity lasted for 1 day. Mean Na intake was not different between the two groups during the 4-day period prior to AmphoB therapy. Mean Na intake during the first 10-day period of AmphoB therapy was significantly lower in the CL group (3.7 vs 6.2; P < 0.001). CONCLUSIONS High Na intake was associated with a reduction in the incidence of AmphoB-induced nephrotoxicity in extremely premature infants with birth weight <1250 gm. We recommend the use of a high Na intake of >4 mEq/kg per day for extremely premature infants during Amphotericin B therapy.