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Aim: The purpose of this study was to investigate the effects of different pretreatment protocols along with different bonding agents on the microleakage of a fissure sealant material. Method: A total of 144 freshly extracted noncarious human third molars were used. The teeth were randomly assigned into three groups with respect to the pretreatment protocol employed: A. Air Abrasion B. Er,Cr:YSGG laser C. No pretreatment (Control). In each group specimens were further subjected to one of the following procedures before application of the sealant: 1. %36 Phosphoric acid-etch (AE) (DeTrey Conditioner 36/ Denstply, UK) 2.AE+Prime&Bond NT (Dentsply,UK) 3.Clearfil S3 Bond (Kuraray, Japan) 4.Clearfil SE Bond (Kuraray, Japan). All teeth were sealed with the same fissure sealant material (Conseal F/ SDI, Australia). Sealed teeth were further subjected to thermocycling, dye penetration test, sectioning and quantitative image analysis. Statistical evaluation of the microleakage data was performed with two way independent ANOVA and multiple comparisons test at p=0.05. For qualitative evaluation 2 samples from each group were examined under Scanning Electron Microscopy. Results: Microleakage was affected by both the type of pretreatment and the subsequent bonding protocols employed (p<0.05). Overall, the highest (Mean=0.36mm) and lowest (Mean=0.06 mm) microleakage values were observed in samples with unpretreated enamel sealed by S3+Conseal F and samples with laser pretreated enamel sealed by Acid Etch+Prime&Bond+Conseal F protocols, respectively (p<0.05). In the acid-etch group samples pretreated with laser yielded in slightly lower microleakage scores when compared with unpretreated samples and samples pretreated with air abrasion but the statistical significance was not important (p=0,179). Similarly, when bonding agent is applied following acid-etching procedure, microleakage scores were not affected from pretreatment protocol (p=0,615) (intact enamel/laser or air-abrasion). For both all-in one and two step self etch adhesive systems, unpretreated samples demonstrated the highest microleakage scores. Conclusions: For the groups in which bonding agent was utilized, pretreatments did not effected microleakage. Both the tested pretreatment protocols and adhesive procedures had different effects on the sealing properties of Conseal F in permanent tooth enamel.