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The viability of periodontal ligament fibroblasts (PDLF) can affect the long-term prognosis of replanted avulsed teeth. When immediate replantation of an avulsed tooth is not possible, the cells should be incubated in a physiological storage medium instantly to maintain their biological activity. The ability of different storage media to preserve PDLF viability has been previously evaluated. However, few studies have showed the effect of temperature on the viability of PDLF cultured with various storage media in vitro.This study was designed to measure PDLF activity by CCK-8 assay to compare the effectiveness at 4, 22 (room temperature), and 37°C under various storage media.Statistical analysis demonstrated that tap water, saline, and saliva decreased cell viability as the storage temperature increased. But the temperature played only a minor role on cell viability when cells were incubated in Hank's balanced salt solution (HBSS), Dubelco's modified Eagle's medium (DMEM), or milk.Within the parameters of this study, it seems that room temperature is adequate for storing the avulsed teeth in HBSS, DMEM, or milk in the extra-alveolar period.