The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB-binding sites per cell detectable after cell breakage and preparation of Triton-insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25-45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3-5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G-actin, that the first (3-5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis.