Hermaphroditism is prevalent in plants but may allow interference between male function (pollen removal and dispersal) and female function (pollen receipt and seed production) within a flower. Temporal or spatial segregation of gender within a hermaphroditic flower may evolve to reduce this interference and enhance male and female reproductive success. We tested this hypothesis using Chamerion angustifolium (Onagraceae), in which pollen removal (male) and pollen deposition (female) were measured directly on hermaphroditic and experimentally produced unisexual flowers. During a single flower visit in the field, bees deposited 159±24 (SE) pollen grains on a stigma and removed 1058±198 grains from each flower. Anther removal did not alter deposition rates. In the laboratory, bees removed 2669±273 pollen grains and deposited 209±72.3 cross-pollen and 120±28.4 facilitated self-pollen grains per visit. The presence of anthers significantly reduced cross-pollen deposition on the stigma. In contrast, pollen removal was not affected by presence of the pistil. These results suggest that within-flower interference affects female function and represents a fitness cost that can be reduced through temporal segregation of gender within the flower.