OBJECTIVE The study reported here was conducted to determine the systemic oral toxicity and to find the no-observed-adverse-effect level of 20 nm positively charged zinc oxide (ZnO(SM20(+))) nanoparticles in Sprague Dawley rats for 90 days. METHODS For the 90-day toxicity study, the high dose was set as 500 mg per kg of body weight (mg/kg) and the middle and low dose were set to 250 mg/kg and 125 mg/kg, respectively. The rats were held for a 14-day recovery period after the last administration, to observe for the persistence or reduction of any toxic effects. A distributional study was also carried out for the systemic distribution of ZnO(SM20(+)) NPs. RESULTS No rats died during the test period. There were no significant clinical changes due to the test article during the experimental period in functional assessment, body weight, food and water consumption, ophthalmological testing, urine analysis, necropsy findings, or organ weights, but salivation was observed immediately after administration in both sexes. The total red blood cell count was increased, and hematocrit, albumin, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration were decreased significantly compared with control in both 500 mg/kg groups. Total protein and albumin levels were decreased significantly in both sexes in the 250 and 500 mg/kg groups. Histopathological studies revealed acinar cell apoptosis in the pancreas, inflammation and edema in stomach mucosa, and retinal atrophy of the eye in the 500 mg/kg group. CONCLUSIONS There were significant parameter changes in terms of anemia in the hematological and blood chemical analyses in the 250 and 500 mg/kg groups. The significant toxic change was observed to be below 125 mg/kg, so the no-observed-adverse-effect level was not determined, but the lowest-observed-adverse-effect level was considered to be 125 mg/kg in both sexes and the target organs were found to be the pancreas, eye, and stomach.