OBJECTIVE To investigate the effect of root canal preparation and residual root filling material on the impedance characteristics of extracted human roots. METHODS Thirty extracted, human single-rooted teeth were mounted in a custom-made apparatus that allowed strict temperature control. Impedance measurements of the roots were made with a file acting as the internal electrode, using a frequency response analyser. The measurements were made under three canal conditions: (i) before chemo-mechanical preparation; (ii) after chemo-mechanical preparation; (iii) after root filling removal to re-establish patency (following placement of root filling). The measurements were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mm coronal to the apical terminus and also at 0.5 and 1 mm past the apical terminus. Impedance values were viewed using Nyquist plots and comparisons made within each tooth, between measurement points along the length of the canal, as well as under the different canal conditions. Equivalent circuits were modelled for different test conditions. RESULTS The impedance decreased from the coronal to the apical levels in all canal conditions in a characteristic way, with an exaggerated drop at the apical terminus. Impedance decreased after chemo-mechanical preparation, but gave higher values compared with before or after instrumentation, once canal filling had been removed. Equivalent circuits remained consistent at the tested positions within the canal, regardless of canal condition, but the circuit component values changed with the impedance. CONCLUSIONS Impedance was influenced by corono-apical position, chemo-mechanical preparation and residual root canal filling material.