The aim of this study was to evaluate: 1) the effect of five dentin desensitizing agents (DDAs) on permeability using hydraulic conductance, and 2) morphological tubule changes with scanning electron microscopy (SEM). The agents tested were Seal & Protect, Gluma Desensitizer, HurriSeal, D/Sense 2, and Super Seal. Thirty extracted human molars were sectioned into 1-mm mid-coronal dentin disks. Dentin permeability was measured at baseline and after treatment using bovine serum and phosphate-buffered saline at 10 psi. Treatments were applied to the occlusal surfaces of dentin according to the manufacturer's instructions. Representatives from each group were selected for SEM observation. Kruskal-Wallis ANOVA and Tukey's post-hoc comparisons were used to evaluate differences between groups. Mean percent reduction in dentin permeability for each group: SuperSeal = 97.5 ± 4.0, HurriSeal = 54.2 ± 35.3, D/Sense 2 = 46.6 ± 20.4, Gluma Desensitizer = 39.6 ± 26.7, and Seal & Protect = 33.8±19.4. The data provided strong evidence of differences in permeability reduction among the agents (p 0.01). Pairwise comparisons of means demonstrated that the effects of SuperSeal differed significantly from the reductions achieved using Seal & Protect, Gluma Desensitizer, and D/Sense 2. Differences in the degree and content of dentinal tubule occlusion were seen among all DDAs under SEM. Of the materials included in this in vitro study, SuperSeal may be the most beneficial when treating dentin sensitivity. The wide range of results may reflect the various approaches and chemistries used to occlude tubules.