The purpose of this study was to evaluate the effectiveness of fissurotomy and double application time of acidic primer of self-etching adhesive system of acid treatment on enamel surfaces for prevention of microleakage of pit and fissure sealants. The microleakage of pit and fissure sealants was evaluated by measuring the penetration depth of methylene blue solution. Specimens were divided by 4 groups according to the method of treatment. Group I: 35% phosphoric acid etching, 20 seconds. Group II: Priming with self-etching primer, 20 seconds. Group III: Priming with self-etching primer, 40 seconds. Group IV: Priming with self-etching primer, 20 seconds, after fissurotomy. The etched pattern produced on enamel was observed using a scanning electron microscope. Obtained data were analysed statistically using Kruscal-Wallis test followed by Mann-Whitney test for comparison of groups. The results are as follows: 1. Microleakage scores of group IV priming with self-etching primer 20 seconds after fissurotomy was the lowest. 2. Microleakage scores between group II and group III were not shown significant difference. 3. Enamel for 20 seconds etching with 35% phosphoric acid was observed the most effective etching pattern. And the etching pattern on the fissure enamel with self-etching adhesive 20 seconds after fissurotomy was more prominent than group II, III only using self-etching primer.