The objective of this study was to evaluate the antimicrobial activity of calcium hydroxide in infected dentinal tubules. Four microorganisms, strains of ATCC (Streptococcus faecalis (ATCC-29212), Staphylococcus aureus (ATCC-6538), Bacillus subtilis (ATCC-6633), and Pseudomonas aeruginosa (ATCC-27853)) and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. Sixty-three human maxillary central incisors were prepared and sterilized by autoclaving. Five groups of 12 teeth each were contaminated for 28 days using new 24-h cultures every 72 h, prepared and adjusted to tube 2 of the MacFarland scale (6 x 10(8) cells/ml). Root canals were then irrigated with 5 ml of saline, dried, and completely filled with calcium hydroxide paste. At intervals of 0, 48, and 72 h, and 7 days, dressings were removed and teeth were immersed in 5 ml of BHI and incubated at 37 degrees C for 48 h to observe the growth and multiplication of the microorganisms. Three uninoculated teeth were maintained in a humid environment as an aseptic control. These teeth were immersed in BHI and maintained at 37 degrees C for 7 days to determine microbial growth. Bacterial growth was shown by turbidity of the culture medium and confirmed by seeding these broths on BHI agar at 37 degrees C for 24 h. The positive BHI tubes were selected, and inoculum was spread on the surface of BHI agar, followed by the same incubation conditions. Gram stain was conducted from BHI growth and from colonies growing on solid medium. Calcium hydroxide in infected dentinal tubules showed no antimicrobial effect on S. faecalis, S. aureus, B. subtilis, P. aeruginosa, or on the bacterial mixture used throughout the experiment.