Direct acridine orange (AO) staining was used to detect bacteria adherent to intravascular catheters (IVC). Samples from 710 IVC tips were first cultured on blood agar plates by a semiquantitative technique and then independently colored with AO and screened dry at a magnification of x100 for 3 min. In the absence of fluorescence, they were considered negative. When fluorescence was present, they were further examined for the presence of microorganisms at x1,000 with immersion oil. Of 710 IVC tips, 37 (5.2%) were positive upon culture (greater than or equal to 15 colonies) and 673 were negative (640 were sterile and 33 [4.6%] had 1 to 14 colonies). The AO sensitivity was 84%, and the AO specificity was 99%. When restricted to the 212 long IVC, AO sensitivity rose to 94%. AO staining was positive in all cases of catheter-associated bacteremia. The negative predictive value of the preliminary screening at x100 was 99.5%. The direct examination of IVC tips stained by AO appears to be a simple and rapid method for diagnosing IVC-associated infections. In addition, AO staining is easier to perform than Gram staining.