Background: In neonates, the acridine orange leukocyte cytospin (AOLC) test has been found to be a highly sensitive test for the detection of infected IV catheters in situ, which provides a result in less than 1 hour. Preliminary data suggested that the AOLC test was of limited value in adults. We report here a modification of the test for adult patients with indwelling central venous catheters. Methods: A prospective study was performed on two groups of 50 adult patients with suspected sepsis and a central venous catheter. The AOLC test was carried out after the clinical decision to remove the catheter had been made. In group 1 patients, a blood sample was withdrawn from the catheter for the AOLC test. In the patients in group 2, an endoluminal brush was used to 'sweep' the catheter before the collection of the blood sample. Results of the AOLC test were compared with culture of the removed catheter tip. Results: From the catheters in group 1 (no brush), 17 catheter tips were found to be infected, but the AOLC was positive in only two patients (12%). In group 2 (brush), 18 tips were infected, and the AOLC test was positive in 15 patients (83%). The use of the endoluminal brush significantly improved the yield of the AOLC test (p < .01) to levels reported in neonates. The AOLC test, produced no false positives in either group. Conclusion: When used independently, the AOLC test was not sensitive enough to detect catheter-related sepsis. However, in combination with an endoluminal brush, the AOLC test was much more sensitive and has the potential to provide a simple, rapid, and accurate diagnostic test for catheter-related sepsis, which does not require removal of the catheter.