The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)(2)) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degreesC in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)(2) USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)(2) USP (0-1 g/ml H2O); group 3, Pulpdent TempCanal(TM) paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)(2) can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)(2) may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.