Objective: Dental pulp has been used as a source in various stem cells experiments in the recent years. The aim of this study was to isolate dental pulp stem cells in sterilized conditions, to define the content level of stem cells and to optimize the methods that were used in obtaining single cell suspension for cell culture experiments. Material and Methods: In this study, a third molar tooth was used. Dental pulp tissue was digested with collagenase Type 1 (3 mg/mL) in alpha-MEM containing 10% fetal calf serum to obtain single cell suspension. The cells were incubated in 6-well-plates with culture medium at 5% CO(2) for 72 hours at moist conditions. After 72 hours, the cells were examined by inverted microscopy for cell morphology and development of colonies and the ratio of CD34 positive cells were defined with flow cytometry. Results: The development of the cell colonies was observed in culture. Three different cell populations were defined from the culture and the ratios of these populations that expressed CD34 were 10.99%, 12.99% and 2.99%. Conclusion: Considering the high rate of CD34 positive cells and the capability of forming colonies in in vitro culture, dental pulp is a good stem cell source.