Introduction: the aim of this in vitro study was to evaluate whether an apical negative pressure irrigation system was more effective in the removal of smear layer and organic debris from the root canals than conventional irrigation system with syringe. Methods: 20 single-rooted extracted human teeth were selected by radiographs taken in both the mesiodistal and buccolingual directions. After shaping with nickel-titanium instruments to a master apical file size #40, specimens were devided in two groups according to the root canal irrigation system. Teeth assigned to group 1 (n=10) received a final irrigation with conventional syringe and a 30-gauge needle (NaviTip; Ultradent, South Jordan, UT); teeth assigned to group 2 (n=10) were rinsed with an irrigation device based on negative apical pressure (EdoVac, Discus Dental, Culver City, CA).The amount of residual smear layer and debris was evaluated at the scannig electron microscope analysis, and a semi-qualitative score was assigned to each tooth at the coronal, medium and apical third of the root canals. A nonparametric test was performed to compare the results of the S.E.M. analysis. Results: no significant differences (p>0.05) were found between the two irrigation methods in the removal of smear layer in the whole lenght of the root canal walls. Apical negative pressure removes statistically more debris (p=0.022) than conventional syringe in the apical third of the root canals, but no statistical differences were found between group 1 and 2 in the medium and coronal part of the samples. Conclusions: the outcome of this study indicates that the EndoVac system removes more debris in the apical region of the root canal than the conventional needle irrigation, but there are no significant differences in the removal of smear layer between the two techniques.