The most important aim in periodontal therapy is to regenerate the periodontal supporting tissue lost as a result of periodontitis. Several studies have demonstrated that tobacco use interferes with periodontal therapy and substantially reduces the possibility of favorable treatment outcomes. The present study was performed to determine the attachment of PDL cells to the diseased roots of a smoking patient compared to non-smoking controls with enamel matrix derivative (EMD) application. Teeth both from a patient smoking more than 20 cigarettes daily and from another non-smoking patient were extracted and PDL tissue biopsies were taken from these teeth. Fibroblasts were cultured. Each root surface was divided into six equal parts. Samples were treated with citric acid and EMD, embedded into cell culture flasks, and kept in the culture for 1 h, 3 h, 5 h and 3 days. Then, electron microscopy analysis was performed. In the smoking group, collagen fibers were spread parallel to the surface as in the non-smoking group, but in one single direction rather than in different directions. It was observed that EMD application on smoking and non-smoking periodontally-diseased patients could affect the function of PDL cells and the potential of collagen production.