Objective: Anti-nuclear antibodies (ANA) develop against the structures found in the cell nucleus. These antibodies can be positive in the autoimmune disorders, but they can be also detected in healthy people. The objective of our study was to determine the definitive diagnosis of the patients referred to our clinic due to the ANA positivity and find out whether they develop rheumatologic disorders during the clinical follow-up. Material and Methods: We have reviewed the medical files of children who were referred to the pediatric rheumatology department between 2014 and 2016 with ANA positivity. Results: 43 subjects were enrolled in the study. The complaints of the referred patients at first presentation were as follows: joint symptoms in 19 patients (44.2%), mucocutaneous symptoms in 13 patients (30.2%), hematological findings in 6 patients (14%), neurological symptoms in 3 patients (7%), and Raynaud’s phenomenon in 2 patients (4.6%). 34 patients (79%) had a positive ANA titer ≥ 1/160. The ANA titer level was below 1/160 in 9 patients (21%). 23 patients (53.4%) were diagnosed with a rheumatologic disease, while 20 patients did not have any rheumatologic disorder. There was no significant difference between the ANA-positive (n=34) and ANA-negative (n=9) patients with regards to the clinical and laboratory characteristics. The comparison of the patients with and without a rheumatologic disorder revealed that the presence of auto-antibodies was more common and acute phase reactant levels were higher in the disease group for arthralgia, arthritis and Raynaud’s phenomenon. Conclusion: We conclude that ANA testing should preferably be requested in the presence of clinical findings associated with rheumatologic disorders.