This study examined the mechanism governing the occurrence of defect layers of incisor dentine in Mg-deficient rats by X-ray microanalysis. Young (5 weeks of age) Wistar male rats were pair-fed semi-synthetic diets containing either control (0.05% Mg) (N = 8) or Mg-deficient (0.001% Mg) (N = 8) ingredients for 17 days. All animals were time marked with a combination of 0.1 mol nitrilotriacetato lead and 0.1 mol nitrilotriacetato zinc (2mg Pb/kg body weight) on days 0, 7 and 14 into incisor dentine. Blood samples were obtained on days 10 and 17 in order to measure Ca, Mg, P and alkaline phosphatase activity levels in serum; moreover, hypomagnesaemia and hypercalcaemia were confirmed. After the 17th day, rats were sacrificed humanely under anaesthesia and mandibular incisors were removed. Dentine formation of right mandibular incisors was assessed (time marking lines); furthermore, Ca, P, Mg and sulphur (S) concentrations as well as Ca/P molar ratio were determined in left mandibular incisors based on contiguous measurement points at 2 microm intervals from dentine pulp to dentine of the lingual aspect via X-ray analysis. Additionally, proteoglycan distribution was observed in other Mg-deficient rat dentine. These findings demonstrated decreases in body weight, incisor formation and incisor length in Mg-deficient rats. Mg and S levels increased in the defect layers, whereas proteoglycan decreased. This phenomenon was possibly attributable to condensation of Mg and S contents consequent to decreased dentine formation during Mg-deficiency and a transient increase in Mg due to transport of Mg as a result of inhibition of cell proliferation in soft tissues.