The cis-acting elements for regulating gene expression of the tobacco pathogenesis-related 1a protein gene were analyzed in transgenic plants. The 5'-flanking 2.4-kilobase fragment from the pathogenesis-related 1a protein gene was joined to the bacterial beta-glucuronidase gene and introduced into tobacco cells by Agrobacterium-mediated gene transfer. Promoter activity was monitored by quantitative and histochemical assay of beta-glucuronidase activity in leaves of regenerated transgenic plants. The level of beta-glucuronidase activity was clearly increased by treatment with salicylic acid, by cutting stress, and by local lesion formation caused by tobacco mosaic virus infection. Cytochemical studies of the induced beta-glucuronidase activity revealed tissue-specific and developmentally regulated expression of the pathogenesis-related 1a gene after stress or chemical treatment and after pathogen attack. To identify the cis-acting element more precisely, a series of 5'-deleted chimeric genes was constructed and transformed into tobacco plants. Transgenic plants with a 0.3-kilobase fragment of the 5'-flanking region of the pathogenesis-related 1a gene had the same qualitative response as those with the 2.4-kilobase fragment upon treatment with salicylic acid or infection with TMV. Thus, the 0.3-kilobase DNA sequence fragment was sufficient to allow the regulated expression of the pathogenesis-related 1a gene.