Purpose To investigate the pattern of antibiotic use in five Chinese children's hospitals from 2002 to 2006. To see if the Guidelines to encourage rational use of antibiotics issued by the Ministry of Health in October 2004 have any impact on the use. Methods The Anatomical Therapeutic Chemical Classification/Defined Daily Doses (ATC/DDD) methodology was used. Aggregate data on antibiotic use (ATC code-J01) were expressed in numbers of DDD/100 bed-days for inpatients. Results Total 56 different substances of systemic antibiotics were used. The overall consumption of antibiotic drugs was 68.2, 58.4, 65.8, 65.6 and 49.9 DDD/100 bed-days for the years 2002-2006, respectively. The top antibiotics used were third-generation cephalosporins. There was considerable variation in both type and amount of antibiotics used in the five hospitals. In 2002, some hospitals had twice the antibiotic use compared to others. While the overall antibiotic use in 2005 was largely unchanged compared with previous years, by 2006 antibiotic use had decreased by 22.6% and the variation in use between hospitals was also reduced. Conclusions The ATC/DDD methodology proved useful for studying overall antibiotic usage in children's hospitals. The decline in antibiotic usage found in 2006 (and the reduced variation between hospitals) may be attributed to the impact of the Ministry of Health guidelines which took some time to be promulgated to individual staff members. Further research will focus on compliance of antibiotic use in these five hospitals with particular guideline recommendations for specific clinical problems such as bacterial resistance and surgical antibiotic prophylaxis. Copyright (c) 2007 John Wiley & Sons, Ltd.