The aim of this study was to examine the effect of fluoride application on 37% phosphoric acid-etching by atomic force microscopy (AFM) in primary tooth samples based on a clinical protocol used in a pediatric dental hospital. Enamel samples were prepared from 36 exfoliated and non-carious primary teeth. Primary tooth samples were randomly assigned to one of the four groups based on the timing of acidetching with 37% phosphoric acid after an acidulated phosphate fluoride (APF) pre-treatment. Group 1 received no fluoride application, Group 2 was pre-treated with fluoride and then received acid-etching 2 weeks later. One week separated the fluoride treatment and the acid-etching in Group 3, while Group 4 received acid-etching immediately after the fluoride treatment. The vestibular enamel surfaces of each primary tooth sample were scanned in air at a resolution of 512 ร 512 pixels and a scan speed of 0.8 line/ s. On the enamel surfaces of the primary teeth after APF pre-treatment, debris were observed although the teeth were smoother than they were prior to APF. As a result, it was concluded that APF treatment is responsible for decreased primary tooth surface roughness. The enamel surfaces etched for 20 s showed that acid-etching was effective not only in removing scratches and debris, but also for evaluating enamel rod characteristics. Primary tooth enamel surfaces after etching showed minute structures caused by the decreased hydroxyapatite nanoparticle space, compared to those before etching. Also, acid-etching showed significantly increased roughness effects (p < 0.0001, n = 9). Finally, as more time elapsed after APF pre-treatment, the roughness was decreased to a lesser degree (p = 0.005, n = 9). We suggest that primary teeth etching 2 weeks after APF pre-treatment used clinically in pediatric hospitals may be effective to obtain properly etched enamel surfaces.