In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved in the control of cell motility and chemotaxis, monoclonal antibodies were raised against proteins that had been enriched on an F-actin affinity matrix. One antibody recognized a protein distinguished by its strong accumulation at the tips of filopods. These cell-surface extensions containing a core of bundled actin filaments are rapidly protruded and retracted by cells in the growth-phase stage. The protein of 269 kD turned out to resemble mouse fibroblast talin (Rees et al., 1990) in its primary structure. The fit is best among the first 400-amino acid residues of the NH2-terminal region where identity between the two proteins is 44% and the last 200-amino acid residues of the COOH-terminal region with 36% identity. In the elongated cells of the aggregation stage the Dictyostelium talin is accumulated at the entire front where also F-actin is enriched. Since this protein exists in a soluble state in the cytoplasm, mechanisms are predicted that cause accumulation at sites of the cell where a front is established. Evidence for receptor-mediated accumulation was obtained by local stimulation of cells with cAMP. When a new front was induced by the chemoattractant, the talin accumulated there within half a minute, indicating a signal cascade in Dictyostelium responsible for assembly of the talin beneath sites of the plasma membrane where chemoattractant receptors are strongly activated. The ordered assembly of the talin homologue together with actin and a series of other proteins is considered to play a key role in chemotactic orientation.