The self-etching adhesive has an advantage of quick application in young children. Objective: The present study compared the effects of phosphoric acid and self-etching primers on the microleakage and penetration ability of a sealant. Materials and Methods: Thirty extracted non-carious human molars were randomly divided into 3 groups (10 teeth each). The teeth were etched [group 1 with 37% phosphoric acid for 15 s and rinsed with water, group 2 with Clearfil SE Bond primer (Kuraray Medical Inc., Japan) for 20s and group 3 with AdheSe primer (Ivoclar/Vivadent, Liechtenstein) for 30s] and air-dried prior to the application of a pit and fissure sealant (Prevocare, Thailand). The teeth were subjected to thermocycling (5000 cycles, 5 and 55°C) and stored in 5% methylene-blue solution for 24h. They were vertically sectioned (1mm thick) and only V-shaped fissures were examined using stereomicroscope at 20X magnification. Microleakage (as % dye penetration distance to total interface distance) and the penetration ability (as % sealant penetrating depth to total fissure depth) were determined. Results: There was no significant difference among the penetration ability of the three groups (Kruskal-Wallis test, p>0.05). However, the AdheSe group showed significantly more leakage compared to the other two groups (ANOVA with Bonferroni test, p<0.05). Conclusion: Clearfil SE Bond self-etching primer may be an alternative to the phosphoric acid-etch technique for sealant application.