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β-Glucuronidase is a lysosomal enzyme released into the extracellular fluid during inflammation. Increased β-glucuronidase activity in the cerebrospinal and peritoneal fluid has been shown to be a useful marker of bacterial inflammation. We explored the role of β-glucuronidase in the detection of bacterial infection in bronchoalveolar lavage fluid (BALF) of paediatric patients.In this case–control study, % polymorphonuclear cell count (PMN%), β-glucuronidase activity, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and elastase were measured in culture-positive (≥104 cfu/mL, C+) and -negative (C−) BALF samples obtained from children.A total of 92 BALF samples were analysed. The median β-glucuronidase activity (measured in nanomoles of 4-methylumbelliferone (4-MU)/mL BALF/h) was 246.4 in C+ (interquartile range: 71.2–751) and 21.9 in C− (4.0–40.8) (P < 0.001). The levels of TNF-α and IL-8 were increased in C+ as compared with C− (5.4 (1.7–12.6) vs 0.7 (0.2–6.2) pg/mL, P < 0.001 and 288 (76–4300) vs 287 (89–1566) pg/mL, P = 0.042, respectively). Elastase level and PMN% did not differ significantly (50 (21–149) vs 26 (15–59) ng/mL, P = 0.051 and 20 (9–40) vs 18 (9–34) %, P = 0.674, respectively). The area under the curve of β-glucuronidase activity (0.856, 95% confidence interval (CI): 0.767–0.920) was higher than that of TNF-α (0.718; 95% CI: 0.614–0.806; P = 0.040), IL-8 (0.623; 95% CI: 0.516–0.722; P = 0.001), elastase (0.645; 95% CI: 0.514–0.761; P = 0.008) and PMN% (0.526; 95 % CI: 0.418–0.632; P < 0.001).This study demonstrates a significant increase of β-glucuronidase activity in BALF of children with culture-positive bacterial inflammation. In our population β-glucuronidase activity showed superior predictive ability for bacterial lung infection than other markers of inflammation.