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Objective: To determine Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) isolated from periodontal patients and healthy subjects using culture and PCR methods. Methods: Duplicate paper point needles were taken from 100 samples (50 healthy subjects and 50 patients), who referred to the specialized dental clinic from Oct. 2015 to Mar. 2016. In laboratory after incubation period and observing the star-shaped colony A. actinomycetemcomitans, the confirmation tests, including gram staining and catalase test were carried out. For PCR, samples were analyzed with genus specific primers. These primers set, amplified a 500 bp fragment. Results: Of the 100 samples, A. actinomycetemcomitans was isolated from 31 patients (31%), (24 isolate of patients, and 7 isolate of healthy subjects) by using a selective Aggregatibacter isolation medium. Using PCR, a total of 49 (49%) samples were found to be positive for A. actinomycetemcomitans (35 isolate of patients, and 14 isolate of healthy subjects). Conclusion: PCR was found to be highly sensitive when genus specific primers were used for diagnosis of A. actinomycetemcomitans in comparison with culture method.