An in vitro study was performed to assess the depth of penetration of a fissure sealant after cleaning the occlusal surfaces with an air-polishing unit, and to compare the results with those obtained after cleaning with pumice and water and after no cleaning. Forty-six clinically non-carious premolars and permanent molars were randomly distributed into three groups. Following cleaning, etching and fissure sealing, the teeth were sectioned. Each section was examined under a light reflecting microscope and the depth of resin penetration was determined. Fissure cleaning with an air-polishing unit produced a statistically significant increase in depth of penetration of sealant resin, and its use as a standard cleaning method before fissure sealing is recommended.