OBJECTIVE Identify risk factors for first-onset healthcare-associated infection (HAI) in a pediatric intensive care unit (PICU). METHODS Prospective cohort study. METHODS Medical-surgical PICU in a hospital for patients in the public healthcare system. METHODS From January 2005 to June 2006, daily surveillance was carried out on 870 patients ages 0 to 18 yrs during their stay in the PICU through to 48 hrs after discharge (5773 patient-days). RESULTS In 256 admissions, there were 363 episodes of HAI, with a cumulative incidence of 41.7% and a density of 62.9 of 1000 patient-days. Intrinsic and extrinsic factors were investigated and measured until occurrence of first-onset HAI (diagnosed according to Nosocomial Infection Surveillance System criteria) or until discharge or death. In the multivariate logistic regression analysis, risk factors for first-onset HAI in the PICU (controlled for length of stay) were as follows: age under 2 years (odds ratio [OR]), 1.80; 95% confidence interval [CI]), 1.30-2.49); days on ventilator duration (OR, 1.16; 95% CI, 1.08-1.25); transfused blood products (OR, 1.49; 95% CI, 1.08-2.06), glucocorticoids (OR, 1.45; 95% CI, 1.04-2.02) and H2 blockers (OR, 1.47; 95% CI, 1.05-2.06). CONCLUSIONS Efforts toward a reduction in the exposure to extrinsic risk factors should be made, as each of these factors separately explains 30% of the risk of HAI. Interventions directed at processes related to the use of a ventilator and limitations on its duration of use should be a priority in HAI control strategies, as each day of ventilator use increases the risk of HAI.