Purpose: To evaluate the effect of two materials, modified applications, moisture contamination and fissure types on the microleakage and penetration ability of fissure sealants. Methods: 140 extracted human molar teeth were randomly assigned to 14 groups. The three following factors were tested: 1) two sealing materials (Concise and Tetric Flow), 2) four modified applications (cleaning with an air polishing device, etching with a vibration system, the use of drying agent and sealant application with a vibration system), 3) the procedures performed under two conditions (at ambient room condition and in a humidity chamber). Each tooth was subjected to thermal cycling (5000 cycles at 5-55°C) and dye immersion (5% Methylene blue for 24 hours). After sectioning, microleakage, penetration ability and fissure type were examined. Multiple regression analyses were used for statistical analysis. Results: The type of sealing materials and the modified applications were significantly associated with microleakage (P<0.001). The significant impacts on penetration ability were the fissure type, material and mode of application (P<0.004).