Pathogenesis-related (PR) proteins form a heterogeneous group of host-encoded, low-molecular-mass proteins that are secreted through the exocytic pathway. They are synthesized by the plant in response to various stimuli, including pathogen attack or exposure to certain chemicals. The PRB-1b gene of Nicotiana tabacum codes for a basic-type PR-1 protein whose transcription is regulated by ethylene. A minimal ethylene-responsive promoter element was defined by deletion analysis in transgenic tobacco plants. Promoter sequences containing 213 bp or more were sufficient to enhance a 20-fold increase of β-glucuronidase reporter gene expression in transgenic tobacco leaves exposed to 20 µl l−1 of ethylene, while 67 bp were not sufficient to trigger ethylene responsiveness. All the constructs that retained ethylene inducibility exhibited phloem-specific activity, which was constitutive in petiole and pedicel abscission zones. This functional study was correlated to an in vitro screening of the major nuclear proteins’ binding sites present on the promoter. Gel-shift analysis using nuclear extracts from ethylene-treated and non-treated plants revealed five sequence-specific protein-DNA complexes on promoter sequences spanning −863 to −142 bp. Constitutive expression of the basic-type PR-1 genes at the leaf and petiole or flower and pedicel interfaces may represent pre-emption of plant defenses against potential pathogens, suggesting a functional similarity to pathogen-induced expression in the leaf.