Introduction: The aim is to compare different methods for the removal of calcium hydroxide (Ca(OH)2) from root canals. Material and methods: 160 extracted human teeth were divided into 2 groups. In group 1 (n = 80), all root canals were prepared with hand instruments to ISO size 40 and in group 2 (n = 80) by rotary nickel-titanium files (Mtwo) to size 04/40. After rinsing, all root canals were filled with Ca(OH)2 and the access cavity was temporized. All teeth were stored for 7 d at 37 °C and 100 % humidity. After storage, in half of the specimens of both groups (n = 40) root canal irrigation without previous instrumentation was performed. In the other half (n = 40) root canals were instrumented to working length with Hedstrom file ISO size 45. All specimens were divided in subgroups (n = 10) and rinsed with 5 ml of NaCl-solution 0.9 %, CHX 2 %, and NaOCl 2.5 % with or without ultrasonic activation, respectively. By scanning electron microscope evaluation the cleanliness of the root canal walls was scored from 1 (no Ca(OH)2 visible) to 5 (pronounced layer of Ca(OH)2). The data obtained were statistically evaluated by Kruskal-Wallis-test (p 0.05). Results: Ultrasonic-activated NaOCl removed significantly more Ca(OH)2 than all other solutions or methods (p 0.05). The instrument taper (hand instruments 2 % or NiTi files 4 %) as well as instrumentation before rinsing, had no significant influence (p > 0.05). For all rinsing solutions tested, the result within the respective group was independent of the localization in the root canal (p > 0.05). Conclusion: Only passive ultrasonic activation was able to remove Ca(OH)2 from the root canal sufficiently. Neither the taper of the instruments used nor instrumentation before rinsing had an significant influence on the removability of Ca(OH)2. Keywords: calcium hydroxide, instrument taper, passive ultrasonic irrigation (PUI), root canal dressing, root canal irrigation