Purpose: To evaluate the effect that fissure depth and enamel conditioning protocols have on penetration abilities of resin-based and glass-ionomer fissure sealants. Materials and Methods: Fifty extracted human molars were randomized into 10 experimental groups. Conditioning was performed as follows: groups I and VI, no pretreatment; groups II and VII, 10% polyacrylic acid and 37% phosphoric acid, respectively; groups III and VIII, self-etching adhesive; groups IV and IX, Er:YAG laser; groups V and X, Er:YAG laser/10% polyacrylic acid and laser/37% phosphoric acid, respectively. In groups I to V, teeth were sealed with a glass-ionomer material, and in groups VI to X a resin-based sealant was used. The sealed teeth were sectioned and the penetration ability was evaluated using scanning electron microscopy (SEM). Results: Pretreatment with a suitable acid resulted in superior penetration of glass-ionomer and resin-based sealant when compared with other conditioning protocols (p 0.0001), except for the teeth treated with both Er:YAG laser and acid. Both conditioning protocol and fissure depth significantly affected the sealant penetration (p 0.0001), but fissure depth had a greater impact on fissure penetration than did conditioning protocol. Conclusion: Resin-based and glass-ionomer sealing materials show similar potential for pit-and-fissure penetration. Penetration of a fissure sealant is largely influenced by the fissure depth. Regarding the conditioning protocol, a pretreatment with a suitable acid is essential to obtain an adequate penetration of a sealing material. Schlagwörter: fissure sealant, unground enamel, conventional adhesive system, self-etching adhesive system, fissure morphology, glass ionomer, resin composites