Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.