The purpose of this study was to find an effective inactivating agent for chlorhexidine that would facilitate removal of all residual antimicrobial effect, which may cause false-negative results during microbiologic culturing.L-alpha-lecithin, Tween 80, and sodium thiosulfate were used in different proportions to prepare 6 potential inactivating solutions. Nine mL of each inactivating solution was mixed with 1 mL of 2% chlorhexidine solution. After 5 minutes of equilibration, 0.1 mL of bacterial cell suspension containing 2 x 10(4) viable cell of Enterococcus faecalis was added to the mixture. At 10 and 60 minutes, 0.1-mL aliquots were withdrawn and spread over blood agar plates and incubated at 37 degrees C for 72 hours. The number of colony-forming units on the blood agar plates was determined and recorded.The combination of 3% Tween 80 and 0.3% L-alpha-lecithin was found to be the most effective inactivating agent, allowing full recovery of the test organisms in the presence of chlorhexidine.The present study demonstrated a method to predictably inactivate chlorhexidine.