ABSTRACT The phytohormone auxin has wide-ranging effects on growth and development. Genetic and physiological approaches implicate auxin flux in determination of floral organ number and patterning. This study uses a novel technique of transiently applying a polar auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), to developing Arabidopsis flowers to further characterize the role of auxin in organogenesis. NPA has marked effects on floral organ number as well as on regional specification in wild-type gynoecia, as defined by morphological and histological landmarks for regional boundaries, as well as tissue-specific reporter lines. NPA’s effects on gynoecium patterning mimic the phenotype of mutations in ETTIN, a member of the auxin response factor family of transcription factors. In addition, application of different concentrations of NPA reveal an increased sensitivity of weak ettin alleles to disruptions in polar auxin transport. In contrast, the defects found in spatula gynoecia are partially rescued by treatment with NPA. A model is proposed suggesting an apical-basal gradient of auxin during gynoecium development. This model provides a mechanism linking ETTIN’s putative transcriptional regulation of auxin-responsive genes to the establishment or elaboration of tissue patterning during gynoecial development.