Objectives To examine the variation in quantity and classes of antibiotics used in all 10 tertiary care neonatal intensive care units (NICUs) in the Netherlands during 2005. Methods We collected data from all tertiary care NICUs in the Netherlands on clinical and demographic characteristics and the type and quantity of systemic antibiotic use [expressed as defined daily doses (DDD)/100 admissions] in 2005. Antibiotics were ranked by volume of DDDs, and those antibiotics which accounted for 90% of the total volume of use [drug utilization (DU) 90%] were noted. Results Antibiotic consumption ranged from 130 to 360 DDD/100 admissions. In total, 9–24 different antibiotics were used, of which 3–10 were in the DU90% segment. Conclusions By comparing antibiotic use in Dutch NICUs we found a considerable variation in the number of different antibiotics used and in the total amount of antibiotic use. Further exploration of the opportunities to reach consensus in antibiotic policy, and to increase attention to antibiotic stewardship, is recommended.