In a randomized double-blind cross-over study on 20 spontaneously breathing, oxygen-dependent preterm infants who had received positive pressure ventilation for respiratory distress syndrome, we tested the hypothesis that the one-way non-rebreathing valves of aerosol spacer devices might impair rather than enhance the delivery of aerosols to small infants by metered dose inhalers (MDI). Ten infants were given 2 doses (200 μg/dose) of MDI albuterol through a neonatal Aerochamber® 4 h apart. At random sequence, one dose was delivered with the non-rebreathing valve of the Aerochamber® in place; for the other dose, the valve had been removed. The experiment was repeated on another ten infants using a different spacer device (Babyhaler®) with or without its one-way inspiratory valve removed. During the first hour following aerosol administration, use of the non-valved spacers was associated with a significantly greater degree of tachycardia in both groups, and also lower transcutaneous carbon dioxide tension in the Aerochamber® group. All infants showed a reduction in respiratory system resistance and an improvement in functional residual capacity following albuterol treatment. In both groups, maximum reduction in respiratory system resistance, recorded 30 min after aerosol delivery, was significantly greater following the use of the non-valved spacers (Aerochamber®: 51.2 ± 3.1% vs. 35.0 ± 2.8 %, P <0.0001; Babyhaler®: 38.8 ± 2.3% vs. 19.2 ± 1.4%, P <0.0001) than following the use of the spacers with a valve. The findings provide indirect evidence supporting our hypothesis and suggest that when the MDI is used to deliver therapeutic aerosols to non-ventilated newborns or small infants, a spacer device without a non-rebreathing valve should be used.