This study was done to assess the influence of the topical application of two different desensitizing agents on dentin permeability and dentinal tubule occlusion. Twenty-one rats provided 84 teeth: 36 for the in vitro and 48 for the in vivo investigation. The following agents were tested: Group 1, 2% potassium nitrate plus 2% sodium fluoride gel; Group 2, 5% sodium fluoride varnish; Group 3, 3% hydroxyethylcellulose gel (control). Cervical cavities were prepared and EDTA was applied to expose the dentinal tubules. After each treatment, Evans blue dye was applied to the teeth. Dentin permeability, scanning electron microscope (SEM) sections, and energy dispersive X-ray (EDX) were analyzed. One-way ANOVA was used to compare the data. There were significant differences (P < 0.05) among groups for dentin permeability, number of tubules/mm(2), tubule area and tubular diameter. Groups 1 and 2 (both in vitro and in vivo) showed open and partially occluded tubules. Group 3 had the most open tubules. EDX revealed similar composition for both experimental conditions. Within the limits of the study, 2% nitrate potassium plus 2% sodium fluoride gel and 5% fluoride varnish decreased the dentin permeability, resulting in partial tubular occlusion.