Objectives: To assess the etching effects of universal adhesives applied actively or inactively on human enamel, using two methods of FESEM specimen preparation (direct and resin replica). Methods: Enamel rectangles of extracted molars were divided into 12 groups (n = 4), according to the adhesive, strategy, and application mode: Scotchbond Universal [Sc], self-etch/inactive [ScSeI], self-etch/active [ScSeA], and etch-and-rinse [ScEr]; All-Bond Universal [Al], self-etch/inactive [AlSeI], self-etch/active [AlSeA], and etch-and-rinse [AlEr]; Prime & Bond Elect [Pb], self-etch/inactive [PbSeI], self-etch/active [PbSeA], and etch-and-rinse [PbEr]; Gaenial Bond [Ga], self-etch/inactive [GaSeI], self-etch/active [GaSeA] and etch-and-rinse [GaEr]. Each adhesive (ER, SEI and SEA) was applied in enamel rectangles from the same tooth and processed using two methods of specimen preparation for FESEM. (a) Direct technique - The enamel specimens were immersed in acetone in a rotator immediately after the application of the adhesive. Specimens were left in acetone for 2h followed by 12h; (b) Replica technique – The adhesive was light-cured as per manufacturer's instructions, followed by a 2mm-thck layer of Opallis Flow flowable composite. The specimens were immersed in 6NHCl for 12h in a rotator to obtain a resin replica of the surface. Specimens were mounted on Al stubs, vacuum-dried, and sputter-coated with gold/palladium. FESEM micrographs were obtained from each specimen at standard magnifications and evaluated by a blind evaluator. Results: The etch-and-rinse strategy resulted in the deepest etching pattern. The inactive application of the self-etch strategy resulted in a poor etching pattern, which was slightly improved with active application. The resin replicas of Pb and Ga displayed droplets. Conclusion: A deep enamel pattern etching was only achieved when the etch-and-rinse strategy was used, despite the slight increase in enamel roughness when adhesives were applied in the self-etch mode with active versus inactive application. The FESEM observations of HEMA-free universal adhesives (Pb, Ga) suggest phase-separation reactions.