The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-beta-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in beta-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the distal portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.