Purpose: The objective was to assess the effect of fissure depth and enamel surface preparation protocols on penetration of resin-based, giomer and glass-ionomer fissure sealants. Materials and Methods: Ninety extracted third molars were assigned into three groups according to the material used. All three groups were subsequently divided depending on tooth preparation: no pretreatment or surface preparation with 10% polyacrylic acid, 37% phosphoric acid, or self-etching adhesive. In addition, Er,Cr:YSGG laser or air abrasion were employed in all subgroups. The penetration was evaluated using scanning electron microscopy. Results: The groups that were pretreated with an appropriate acid resulted in superior penetration of glass-ionomer and resin-based sealants when compared with other surface preparation protocols (p 0.05). The exception was teeth treated with both air abrasion and acid etching. Both preparation protocol and fissure depth significantly affected the sealant penetration (p 0.05), but fissure depth had a greater impact on fissure penetration than did enamel surface pretreatment. Conclusion: All investigated materials exhibit similar properties regarding sealant penetration. Penetration of a fissure sealant is significantly influenced by the fissure depth. In regard to enamel surface preparation protocol, a pretreatment with phosphoric acid in resin-based and polyacrylic acid in glass-ionomers appears to be essential in obtaining the adequate penetration of a sealing material. Keywords: fissure sealant, fissure morphology, glass-ionomer, resin composites, giomers