It has been postulated that evacuation systems used in dentistry could be a source of cross-contamination between patients through backflow of bacteria dislodged from the saliva ejector tubings. The bacterial microflora associated with these systems was characterized using transmission electron microscopy (TEM) and microbiological cultures. The potential for backflow was investigated by a study of pressure differentials in evacuation system tubing and by the presence of bacteria in backflow samples. Evacuation lines were coated with microbial biofilms in which microcolonies of Gram-positive cocci and Gram-negative bacilli predominated, embedded in an extensive polysaccharide matrix. Most bacteria were metabolically active. Occasionally, buccal material such as collagen, fibrin and eukaryotic cell debris was observed. In other experiments, flow reversal was detected several times during saliva ejector use though each of these events was brief (less than 0.1 s). Aspiration of saliva, or occlusion of the mouthpiece opening by the oral mucosa, were the major factors leading to backflow episodes. Bacteria associated with backflow were found in almost 25% assays, with counts ranging from 1-300 cfu/occurrence. The majority of the bacteria isolated from biofilm or backflow samples were staphylococci, micrococci and non-fermentive Gram-negative rods. Pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus were also isolated from backflow fluids. No oral streptococci could be recovered from biofilms in the tubing beyond 15 min from the last saliva ejector use however, suggesting that these species did not survive in the biofilms. These data suggest, although without direct proof of cross-contamination, the possible existence of an infectious risk associated with oral evacuation systems, as potential pathogens may be shed from tubing biofilms following backflow. Even if the risk of cross-contamination between patients is considered to be low, the necessity for regular disinfection of these systems must be stressed, since biofilms can serve as a reservoir for pathogens or harbor potentially infectious material.