K252a and staurosporine, protein kinase inhibitors, induced the outgrowth of extremely long extensions by Dictyostelium cells. Histochemical observations revealed that F-actin was selectively localized in the extensions. Cytochalasin D inhibited formation of the extensions, but nocodazole did not. These findings indicate that the inhibitors altered F-actin-based architecture. During induction of the extensions, a 66-kDa protein whose amount in the Triton-insoluble fraction was increased by the inhibitors was found, though the amounts of F-actin and myosin heavy chain remained constant. Thus, this 66-kDa protein may be involved in the alteration of cytoskeletal architecture observed here. This possibility is discussed in relation to protein phosphorylation. In conclusion, the construction of cytoskeletal architectures resulting from F-actin elongation and bundling is regulated by a protein kinase(s) that is inhibited by K252a and staurosporine.