Objectives:The objective of this study was to evaluate the effectiveness of manual, sonic-air and ultrasonic instrumentation with varying irrigation protocols on removal of the smear layer from root canal walls.Study Design:Sixty extracted single rooted human teeth stored in 0.5% saline were used. Periodontal soft tissues were removed followed by crown separation at the cementoenamel junction (CEJ). All the teeth were randomly divided into three groups. Group I was manually instrumented and irrigated with 5.25% sodium hypochlorite (NaOCl) alone and 17% Ethylenediaminetetraacetic acid (EDTA) alternately, same as sonically instrumented Group II and ultrasonically instrumented Group III. The controls for all groups were irrigated with saline solution.Results:Ultrasonic over the sonic-air and manual technique, and the use of a combination of two different solutions (17% EDTA and 5.25% NaOCl) alternatively yielded better outcome.Conclusions:Ultrasonic, sonic-air and manual instrumentation of the root canal and irrigation with combined solutions is effective in removal of the smear layer from the instrumented walls of the root canal.