OBJECTIVE. The primary objective was to determine the rate of and risk factors for nosocomial primary bloodstream infection (BSI) in pediatric intensive care unit (PICU) patients in order to determine the validity of our previously published findings. The secondary objective was to analyze whether risk factors for primary BSI differed by organism type, particularly whether device use was more strongly associated with BSI due to gram-positive organisms. DESIGN. Prospective cohort study. SETTINGS. St. Louis Children's Hospital, a 235-bed academic tertiary care center with a 28-bed combined medical and surgical PICU. PATIENTS. PICU patients admitted between September 1, 1999, and September 1, 2001. OUTCOME MEASURES. Nosocomial primary BSIs. RESULTS. Of 2,310 patients, 55% were male, and 73% were white. There were 124 episodes of primary BSI in 87 patients (3.8%). Coagulase-negative Staphylococcus organisms were the leading cause of BSI (42 of 124 episodes). The rate of BSI was 9 BSIs/1,000 central venous catheter-days. Multiple logistic regression analysis showed that independent predictors of nosocomial primary BSI included higher number of arterial catheter-days (adjusted odds ratio [aOR], 5.7 per day of arterial catheterization; 95% confidence interval [CI], 3.4-9.8), higher number of packed red blood cell transfusions (aOR, 1.2; 95% CI, 1.1-1.4), and genetic syndrome (aOR, 4.7; 95% CI, 1.8-12). Severity of illness, underlying illnesses, and medications were not independently associated with increased risk of nosocomial BSI. CONCLUSION. Arterial catheter use and packed red blood cell transfusion are potentially modifiable risk factors for nosocomial primary BSI in PICU patients. Genetic syndromes may be markers for unrecognized immune defects that impair host defense against microorganisms.