A prospective study was done to assess the comparative use of acridine orange and Gram stains for detecting false-positive BACTEC blood culture bottles, thereby eliminating unnecessary subcultures. A total of 1049 NR 6A and 7A bottles that were instrument-positive in the BACTEC 660 nonradiometric system, but were Gram stain-negative, had aerobic and anaerobic subcultures as well as an acridine orange stain performed. Only five of 1049 (0.5%) instrument-positive, Gram stain-negative bottles grew microorganisms on subculture. Of these five microorganisms, acridine orange stain detected two. All five microorganisms were assessed not to be clinically significant based on review of the patients' charts. Our data showed that the Gram stain and the acridine orange stain are comparable for detecting false-positive NR 6A and 7A bottles. We conclude that subculture of BACTEC-positive, Gram stain-negative NR 6A and 7A bottles is not necessary.