The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objectives of this study were to evaluate the effectiveness of six different media: culture medium, alpha minimal essential medium (alpha-MEM), milk, Hank's balanced salt solution (HBSS), ViaSpan and conditioned medium (CM) to preserve cultured periodontal ligament fibroblasts (PDLF). Periodontal ligament fibroblasts were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media for 2, 8 and 24 h at 4 degrees C. A control group was incubated with culture medium at 37 degrees C. After incubation, cell viability was determined by trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic capacity (by culturing one cell/well). Storage of PDLF up to 24 h decreased their vitality by only 2%-14%. Vitality of the PDLF after 2, 8 and 24 h was highest when stored in milk or HBSS (91%-97%) and lowest when stored in ViaSpan or CM (82%-93%). PDLF stored for 2-8 h in various media had a mitogenic capacity comparable to the control. However, increasing the storage period to 24 h decreased the mitogenicity of the cells by 3%-39%. The highest mitogenicity was found in PDLF stored in milk or HBSS and the lowest in CM or ViaSpan. The clonogenic capacity of the cells dropped by 38%-71% after 24 h and was the best indicator of the deteriorating effect of long storage. Milk and HBSS were the most effective in preserving the clonogenic capacity. Nevertheless, reduction in the viability, mitogenicity or clonogenic capacity was statistically significant in nearly all the tested media only after 24 h of incubation. In conclusion, HBSS and milk were the most effective media for preserving the viability, mitogenicity and clonogenic capacity after storage for up to 24 h at 4 degrees C.