We identified a tobacco stigma-specific gene, designated STIG1. The STIG1 gene is developmentally regulated and expressed specifically in the stigmatic secretory zone. We used a chimeric STIG1-GUS gene to show that the stigma-specific STIG1 gene expression pattern is controlled primarily at the transcriptional level. We constructed a stigma-specific cytotoxic gene by fusing the STIG1 gene 5' regulatory region with the coding sequence of the Bacillus amyloliquefaciens barnase gene, to assess the role of the stigmatic secretory zone in the pollination process. Pistils of transgenic STIG1-barnase tobacco plants undergo normal development, but lack the stigmatic secretory zone and are female sterile. Pollen grains germinate on the ablated 'stigmatic' surface, but are unable to penetrate the transmitting tissue of the style. Application of stigmatic exudate from wild-type pistils to the ablated surface increases the efficiency of pollen tube germination and growth and restores the capacity of pollen tubes to penetrate the style. Our data demonstrate the importance of the stigmatic secretory zone in the pollination process and provide an approach to identify compounds produced by the stigma that are critical for successful pollination and fertilization to occur.