Purpose: To evaluate, in situ, the penetration of deposits formed within the subsurface of dentin samples treated with desensitizing dentifrices designed to occlude dentin tubules compared to two controls. Materials and Methods: Twenty-eight healthy participants wore left and right intraoral appliances, each retaining four human dentin samples, for two periods of 4 days. Samples were power-brushed, outside the mouth, twice daily with test products (dentifrices containing 8% strontium or 8% arginine) or control (1,450 ppm NaF or water) and subjected to an agitated grapefruit juice acid challenge on days 3 and 4. Eighteen dentin samples were randomly selected from each treatment group and were dry fractured for scanning electron microscopy and energy-dispersive x-ray spectroscopy analysis. Results: The proportion of cross-sectioned dentin tubules with subsurface occlusion (occluded to a mean of 5 ± 2 μm, range: 1 to 9 μm below the surface) for the 8% strontium group on days 1 and 2 (pre-acid) was 82% (SD: 9%, 95% confidence interval [CI] = 78% to 86%) and on days 3 and 4 (post-acid) was 88% (SD: 10%, CI = 83% to 93%). For 8% arginine on days 3 and 4 (post-acid), the proportion was 78% (SD: 8%, CI = 74% to 82%). These results were statistically significant compared to those for controls (P .01). The 8% arginine on days 1 and 2 (pre-acid) and water and control paste on all days revealed no subsurface deposit. Conclusions: Within the limitations of this study, cross-sectional SEM analysis suggested strontium and arginine dentifrices occlude tubules subsurface in dentin compared to negative controls following acid challenge. The desensitizing dentifrices elicit subsurface changes that may potentiate their effects for the management of dentin hypersensitivity, particularly for patients who consume acidic beverages.