To elucidate the mechanism of callose plug formation, we studied the time span taken for pollen tubes of Camellia japonica, from the start of plug formation until the stage of plug ingrowth, using electron microscopy. A cell membrane was detected around the plugs at all stages of plug formation, and a small amount of cytoplasm was present near the plugs except for those in the earliest stage of formation. Plug ingrowth proceeded even in the vacuole region of the tube, without any supply of cytoplasm. The cytoplasm near the plugs contained a number of granules, organelles and other structures, but none of these reacted with the anti-callose antibody, whereas, the plugs were intensely labeled at all stages of formation. The results suggest that the plugs are not formed by a successive supply of callosic granules to the inner layer of the tube wall, but by inward auto-growth of a portion of the callosic inner layer of the tube wall, dependent on preferential callose synthesis.