The aim of this study was to evaluate the acridine orange leukocyte cytospin (AOLC) test for the rapid diagnosis of septicemia caused by central venous catheters (CVCs), without removing the catheter, in a pediatric intensive care unit population. Twenty-six patients admitted in the pediatric intensive care unit of Azienda Ospedaliera "Ospedali Riuniti di Bergamo", Italy, were prospectively evaluated for CVC-related infection. Blood for culture was taken from all patients. Quantitative endoluminal cultures of the removed catheter tip by Cleri's technique and semiquantitative superficial cultures of the hub were performed. Gram staining and an AOLC smear were done according to Kite's technique. Four Staphylococcus CVC-related bloodstream infections were identified. CVC colonization was detected in 8 patients. Four had septicemia (Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Candida glabrata) without CVC involvement. However, Gram staining and the AOLC test were negative in all cases. We conclude that cytocentrifugation and acridine orange staining of blood withdrawn by Kite's method from an in situ catheter, although simple, quick, and inexpensive, did not aid diagnosis in this pediatric population.