Between August and November 1997, a nosocomial outbreak caused by gentamicin-resistant Klebsiella pneumoniae occurred in the Neonatal Intensive Care Unit (NICU) of our hospital. Thirteen neonates became colonized and three of them became infected. Comparison of the isolates by amplified fragment length polymorphism (AFLP) revealed clonal similarity for isolates of eight neonates (homology > 90%). Cultures from environmental specimens were negative for gentamicin-resistant K. pneumoniae. A case-control study was conducted to identify risk factors associated with acquisition of gentamicin-resistant K. pneumoniae. Risk factors were low gestational age and birth weight. These neonates need more care and handling and may therefore, be more at risk of colonization. Length of stay on the NICU was significantly longer for cases, but mean time until colonization (6.3 days) was shorter than the total stay for controls (9.5 days). No single member of the medical or nursing staff was significantly more involved with cases than with controls. The outbreak was stopped by replacing gentamicin by amikacin as the antibiotic of first choice whenever the use of an aminoglycoside antibiotic was indicated.