OBJECTIVE To determine the pathogens associated with fulminant (lethal within 48 hours) late-onset sepsis (occurring after 3 days of age) in a neonatal intensive care unit (NICU) and the frequency of fulminant late-onset sepsis for the most common pathogens. METHODS A retrospective study was conducted of sepsis in infants in a NICU over a 10-year period (1988-1997). RESULTS There were 825 episodes of late-onset sepsis occurring in 536 infants. Thirty-four of 49 (69%; 95% confidence interval [CI]: 55%-82%) cases of fulminant late-onset sepsis were caused by Gram-negative organisms, including Pseudomonas sp., 20 (42%); Escherichia coli, 5 (10%); Enterobacter sp., 4 (8%); and Klebsiella sp., 4 (8%). The frequency of fulminant sepsis was highest for Pseudomonas sp., 20 of 36 (56%; 95% CI: 38%-72%) and lowest for coagulase-negative staphylococci, 4 of 277 (1%; 95%CI: 0%-4%). The very low frequency of fulminant sepsis caused by coagulase-negative staphylococci did not increase during the period when oxacillin was used instead of vancomycin as the empiric antibiotic for Gram-positive organisms. CONCLUSIONS These data suggest that empiric antibiotics selected for treatment of suspected sepsis in infants >3 days old need to effectively treat Gram-negative pathogens, particularly Pseudomonas sp., because these organisms, although less frequent, are strongly associated with fulminant late-onset sepsis in the NICU. Avoiding empiric vancomycin therapy seemed to be a reasonable approach to late-onset sepsis, because of the very low frequency of fulminant sepsis caused by coagulase-negative staphylococci.