Purpose: To evaluate the effect of long-term water storage on the microleakage of a fissure sealant applied with or without different bonding agents. Materials and Methods: Extracted human third molars were randomly assigned into 8 groups (n = 24/each). The occlusal surfaces were sealed with a fluoride fissure sealant material (Helioseal F) after one of the following pretreatments: 1. phosphoric acid etching only; 2. phosphoric acid etching + Single Bond; 3. phosphoric acid etching + Prime & Bond NT; 4. Clearfil SE Bond; 5. FL Bond; 6. One Up Bond F; 7. Prompt L-Pop; 8. Mac Bond II. All specimens were thermocycled (1000X), after which half of them (n = 96) were stored in distilled water for 48 h (group A), and the remaining half (n = 96) for 48 months (group B). The specimens were immersed in 0.5% basic fuchsin solution, sectioned, and digitally photographed. Microleakage was evaluated quantitatively using an open-source image analysis toolkit (ImageJ), and the data were analyzed statistically. Results: Four-year water storage significantly increased the amount of leakage in all test groups (p 0.001). In both the absence and presence of water aging, the etch-and-rinse adhesives yielded the lowest microleakage scores (p 0.001). In the 48-h group, the following ranking was achieved in terms microleakage values: phosphoric acid + Prime & Bond NT = phosphoric acid + Single Bond Prompt L-Pop = phosphoric acid etching only FL Bond Clearfil SE Bond Mac Bond II = One Up Bond F. In the 48-month group, the ranking changed as follows: phosphoric acid + Prime & Bond NT = phosphoric acid + Single Bond Prompt L-Pop = FL Bond Clearfil SE Bond = Mac Bond II = One Up Bond F phosphoric acid etching only. Conclusion: Long-term water storage significantly increased the microleakage of Helioseal F applied alone and with a bonding agent. Regardless of the storage term, the use of etch-and-rinse adhesives resulted in significantly less microleakage compared to that achieved with self-etching adhesives or acid etching alone. The sealants placed without a prior bonding agent showed the greatest amount of leakage after four years. Keywords: microleakage, quantitative, etch-and-rinse adhesives, self-etching adhesives, fissure sealants