Senescence of Eustoma flowers is accelerated by pollination which is accompanied by an increase in ethylene production by flowers. We investigated the influence of pollinating different areas of the stigmatic surface on senescence in the flowers of 6 Eustoma cultivars. Anthers were removed from the flowers whose stigma had not yet opened. After the stigmas opened, freshly collected pollen from anthers of same cultivar were placed on them. The pollination levels on stigma were all area, 1/8 area or none (control). Senescence of flowers was significantly accelerated by all and 1/8 area pollination compared with the control. All area-pollinated flowers wilted significantly earlier than did 1/8 area-pollinated ones in three cultivars. In the remaining three cultivars, senescence of flowers by all area pollination tended to be earlier than that of 1/8 area pollination. In ‘Asuka-no-nami’ flowers, ethylene production of flowers rapidly increased 1 day after all area pollination. However, in flowers with 1/8 area pollination, ethylene production slowly increased for 3 days after pollination. A large number of pollen tubes reached the base of the style 2 days after pollination in the all area-pollinated flowers, but on 3rd day in the 1/8 area-pollinated flowers. All area-pollinated flowers required silver thiosulfate complex (STS) at high concentration to delay senescence induced by pollination, compared with the 1/8 area-pollinated flowers. These results suggest that in Eustoma flowers, senescence that is induced by pollination, depends on the pollinated area of the stigmatic surface which resulted in ethylene production.