For identification of risk factors for bloodstream infection (BSI) among neonatal intensive care unit patients, prospective 6-month studies in three neonatal intensive care units were conducted. BSI was diagnosed in 42 of 376 (11.2%) enrolled infants. Pathogens included coagulase-negative staphylococci, Candida sp., Group B streptococci and Gram-negative species. Patients with BSIs were more likely to die during their neonatal intensive care unit stay than were patients who did not acquire BSIs (6 of 42 vs. 11 of 334, P = 0.007). BSI rate was highest in infants with birth weight <1500 g (relative risk (RR) = 6.8, P<0.001), those treated with H-2 blockers (RR = 4.2, P<0.001) or theophylline (RR = 2.8, P<0.001) and those with admission diagnoses referable to the respiratory tract (RR = 3.7, P<0.001). Infants who developed BSI were more severely ill on admission than other infants (median physiologic stability index 13 vs. 10 (P<0.001) and were of lower gestational age (28 vs. 35 weeks, P<0.001). In logistic regression analysis, risk of ESI was independently associated only with very low birth weight, respiratory admission diagnoses and receipt of H-2 blockers. Risk of isolation of a pathogen from blood culture was independently associated with Broviac, umbilical vein or peripheral venous catheterization >10, 7 or 3 days, respectively, at one insertion site. Rate of isolation of a pathogen was higher (9 of 59 (15%)) within 48 hours of a measurable serum interleukin 6 concentration than an interleukin 6 level of 0 pg/ml (10 of 159 (6%), P = 0.04). Conversely >1 day of exposure to gentamicin or ampicillin before the sepsis evaluation was associated with lower BSI risk in infants with intravascular catheters (20 of 127 (16%) vs. 9 of 16 (56%), P = 0.06). These findings indicate that very low birth weight, respiratory diagnoses, H-2 blocker use and prolonged intravascular catheterization at one insertion site are associated with elevated risk of BSI. Clinical trials of interventions addressing these risk factors are warranted.