We studied the in vivo changes of F-actin cytoskeleton in pollen tubes after compatible and incompatible pollinations in Nicotiana alata. The integrity of actin filaments is essential for polar growth in pollen tubes. F-actin provides the track along which organelle and vesicle move, carrying the necessary material to sustain the pollen tip growth. In a previous work we have shown that incompatible pollen tubes underwent gradual depolymerization which continued for several days after complete cessation of pollen growth. To assess whether F-actin fragmentation was an early event in pollen rejection, double labeling of F-actin and the vacuolar compartments was performed in incompatible pollination. Vacuolar breakdown was reported as the crucial step in pollen rejection in Nicotiana because it releases cytotoxic S-RNase to the cytoplasm. At 1 and 3 days after pollination, pollen tubes with depolymerized F-actin were 45% and 70%, respectively. However, less than 10% showed disorganized both F-actin and vacuolar compartments. At 8 d after pollination 90% of pollen tubes had disorganized the F-actin but only 33% displayed vacuolar rupture. No pollen tubes with intact F-actin and disorganized vacuolar compartments were seen at any time. These results suggest that F-actin depolymerization occurs upstream in pollen rejection and precedes S-RNase releasing from vacuole to cytoplasm.