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The present study is aimed to determine serum and urine interleukin-8 (IL-8) levels in premature infants with late onset sepsis (LOS) and to evaluate if urine IL-8 is a useful test for LOS diagnosis. Fifty-six premature infants admitted to the NICU over 1 year had serum and urine IL-8 determined by ELISA. They were divided into three groups: I definite sepsis, II probable sepsis and III non-infected. Results were expressed as mean or median. Differences between groups were assessed by ANOVA, Kruskal-Wallis ANOVA and Dunn's Method. Sensitivity, specificity and positive and negative predictive values were calculated and a receiver operator characteristic curve was constructed to determine serum and urine IL-8 accuracy. There were no differences between groups for birth weight, and gestational and post-natal age. Median serum and urine IL-8 levels were significantly higher in GI and GII: 929 × 906 × 625 pg/ml; P=0.024, and 249 × 189 × 42 pg/mgCr; P < 0.001. Optimal cut-off point was 625 pg/ml for serum IL-8 with 69% sensitivity and 75 pg/mgCr for urine IL-8 with 92% sensitivity. IL-8 can be determined in urine from premature infants with LOS and is an accurate and feasible diagnosis method.