OBJECTIVES: To test the hypothesis that etching enamel with 37% phosphoric acid for 30 s does not lead to detectable mineral loss when measured with transverse microradiography (TMR). DESIGN: An in vitro laboratory investigation. EXPERIMENTAL VARIABLE: Forty bovine incisors were used in the experiment. The crowns of the teeth were covered with acid resistant varnish except for a rectangular area on the labial surface approximately 10 x 12 mm. On the exposed labial surface of 20 teeth an enamel lesion similar to that used in the in situ caries model was induced. Twenty teeth were left without a lesion. The exposed area was divided into three areas of equal size. The control area (C) was covered with acid resistant varnish throughout the experiment. The first experimental area (E1) was etched with 37% phosphoric acid for 30 s and a simulated bracket was bonded to the surface with composite resin. The second experimental area (E2) was left exposed for the remainder of the experiment. The teeth were placed in a demineralizing solution for 24, 48, 72 or 96 h to replicate different cariogenic challenges. OUTCOME MEASURE: Mineral loss as measured with TMR. RESULTS: There were no significant differences in the mineral loss between etched (E1) and etched (C) areas of enamel. There were significant differences in mineral loss between E1 and E2 for the 48 h (p < 0.001) and 72 h (p = 0.001) exposures without a pre-formed enamel lesion. CONCLUSION: There is no detectable mineral loss with TMR when enamel has been etched for 37% phosphoric acid for 30 s. The use of in situ enamel specimens with acid etch retained simulated brackets to investigate demineralization during orthodontics will not significantly affect the outcome compared with unetched specimens