BACKGROUND: Calfactant is an exogenous surfactant used to treat and prevent respiratory distress syndrome in the newborn infant. It is available in single-use preservative-free vials, but contains enough volume to provide multiple doses to small infants. OBJECTIVE: To measure the preservation of calfactant sterility at 12- and 24-hour intervals following initial violation of vial contents and to extrapolate cost savings associated with product conservation. METHODS: A prospective sterility study was performed using calfactant suspension obtained from vials prescribed for infants who had received their dose in the delivery room or the neonatal intensive care unit (NICU). After initial vial entry, test vials were stored in the NICU pharmacy satellite under refrigeration (temperature range 2.2–7.2 °C). Re-entry of test vials and sample removal was performed 12 and 24 hours after initial entry with an 18- or 20-gauge needle. All samples were removed by a neonatal respiratory therapist and placed into 3-mL latex-free, Leurlok plastic syringes and examined by 3 culture media: MacConkey agar, blood agar, and thioglycollate broth. RESULTS: A total of 89 surfactant samples from 45 vials were cultured; 45 at a mean time of 13.36 hours (range 11–41) and 44 at a mean time of 25.8 hours (range 22–60) after initial vial entry. These samples represented the technique of multiple respiratory therapists. All samples were negative for bacterial growth at 24 and 48 hours (β = 0.9). CONCLUSIONS: Results from this short-term sterility study represent an initial step in the evaluation of multiple doses of surfactant from a single-use vial. The data suggest that 1–2 re-entries into a vial of calfactant, within 24 hours after the initial breach, can be a safe and economical method of providing more than a single dose of surfactant to infants weighing <1 kg. We encourage each institution to reproduce these findings before applying this concept to their patients.