Abstract –  Many solutions have been examined as possible storage media for avulsed teeth. The purpose of this study was to compare the effectiveness of several storage media to preserve cultured periodontal ligament fibroblasts (PDLF) under different temperatures. The media tested were: sterile Hank’s balanced salt solution (sHBSS), non-sterile HBSS (nHBSS), skimmed milk, Save-A-Tooth®, Minimum Essential Medium (MEM) and water (negative control). MEM at 37°C was used as positive control. PDLF were obtained from explants of extracted healthy human teeth. Plates containing confluent PDLF were soaked in the various media for 3, 6, 24, 48 and 72 h at 37°C and 20°C. After incubation, viability of the cells was determined using the tetrazolium salt-based colorimetric (MTT) assay and the Trypan Blue exclusion test after 6, 24, 48 and 72 h of incubation at 20°C. The results were analyzed statistically using Kruskal–Wallis, Scheffé and Mann–Whitney (α = 5%) tests. Results from the MTT assay at 37°C and 20°C showed that skimmed milk was the best storage medium for up to 24 and 48 h, respectively, followed by nHBSS and sHBSS. Results from the Trypan Blue exclusion test showed that the best storage media were milk, sHBSS and nHBSS, with no statistical differences, for any time period. The Save-A-Tooth® had a detrimental effect on cells after 24 h. The influence of temperature on the effectiveness of the storage media tested showed at 20°C a decreasing order of efficacy as follows: milk > sHBSS and nHBSS > MEM > Save-A-Tooth® > water while at 37°C it was: MEM > nHBSS > milk > sHBSS > Save-A-Tooth® > water. In conclusion, incubation temperature altered the effectiveness of the storage media and skimmed milk at 20°C was better than HBSS in maintaining PDLF viability.