This study analyzed the efficacy of autologous platelet-rich fibrin (PRF) in maintaining and recovering cell viability of the periodontal ligament (PDL). The PDL cells were isolated from 45 extracted teeth randomly distributed among 6 groups: 5 min, 1 h, 2 h, PRF 30 min, PRF 1 h and PRF 2 h. In the groups 5 min, 1 h and 2 h (n = 5), the teeth were kept dry in extra-alveolar times of 5 min, 1 h and 2 h respectively. The teeth of the groups PRF 30 min, PRF 1 h and PRF 2 h (n = 10) were kept dry at extra-alveolar times of 30 min, 1 and 2 h followed by immersion in PRF for 45 min. PDL cells were isolated by enzymatic digestion with type II collagenase and dispase, counted and analyzed for viability with Trypan blue vital dye in Neubauer chamber. The variables total number of cells and cell viability demonstrated that in the 5 min, 1 h and 2 h groups there was a decrease after the extra-alveolar dry times of 1 and 2 h. In comparison with the total number of cells, group 1 h, considered immediate reimplantation, did not present statistical difference when compared to the groups PRF 30 min, PRF 1 h and 2 h, a result that demonstrates that PRF assists in cell maintenance and recovery. PRF provided increased cell viability in relation to the different dry extra-alveolar times analyzed (p < 0.001). Autologous PRF presented effectiveness in maintaining and recovering PDL cells from extracted teeth and kept dry for up to 2 h.