We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.