Prezygotic interspecific incompatibility is the active recognition and rejection of pollen or pollen tubes from interspecific crosses. Successful interspecific pollinations result in hybrid plants with unique gene combinations. However, interspecific fertilization frequently wastes parental resources due to seed abortion or unhealthy hybrid offspring. The ability of plants to identify and prevent interspecific pollinations preserves reproductive resources for successful intra-specific pollinations. Mechanisms of interspecific incompatibility are diverse and the factors regulating them have not been fully identified. This research identifies interspecific incompatibility mechanisms that result in Nicotiana obtusifolia pollen being rejected by the N. tabacum style. A transgenic N. tabacum plant was developed with a transmitting tract (TT)-ablated style that had no mature TT cells. Pollen tube growth of N. obtusifolia and N. repanda was promoted in TT-ablated style relative to growth in the normal style. This reversal of incompatibility demonstrated that the mature TT was required for interspecific incompatibility. The TT-ablated style was used to develop a novel pollen tube growth assay (PTGA). Ablation of the TT produces a hollow style that is amendable to liquids being injected into the style prior to pollination. Injection of control medium had no effect on pollen tube growth of N. tabacum or N. obtusifolia in the TT-ablated style. Through the application of this novel PTGA, proteins from the N. tabacum TT that regulate the interspecific incompatibility interaction were purified and identified. Proteins extracted from the normal N. tabacum TT specifically inhibited pollen tube growth of N. obtusifolia and N. repanda when injected into the TT ablated style prior to pollination. The proteins required for inhibition were sequenced and identified using tandem mass spectrometry. The major protein component of the active fraction was a class III pistil extensin-like protein (PELPIII), indicating a function in interspecific incompatibility. To complement the PTGA experiments, PELPIII antisense N. tabacum plants were tested for compatibility with N. obtusifolia and N. repanda pollen. Plants with undetectable PELPIII protein had compatible growth of N. obtusifolia and N. repanda. The loss of interspecific inhibition in PELPIII antisense styles further supported that PELPIII was required for interspecific incompatibility in the N. tabacum style.