Appressoria are essential penetration structures for many phytopathogenic fungi. Here F-actin localization dynamics were documented during appressorium formation in vitro and in planta in Colletotrichum graminicola Four discernible stages of dynamic F-actin distribution occurring in a programmed order were documented from differentiation of appressoria to formation of penetration pores: (stage A) from germ tube enlargement to complete expansion of the appressorium; (stage S) septation occurs; (stage L) a long period of low F-actin activity; (stage P) the penetration pore forms. The F-actin subcellular localization corresponded to each stage. A distinct redistribution of actin cables occurred at the transition from stage A to stage S. The in planta assays revealed that F-actin also assembled in invasive hyphae and that actin cables might play an essential role for penetration-peg development. The F-actin localization distribution may be used as a subcellular marker to define the developmental stages during appressorium formation.