Phytochrome represses transcription of its own phyA genes within 5 min of light-triggered conversion to its active Pfr form. We have utilized microprojectile mediated gene transfer into etiolated rice seedlings to delineate sequence elements in the oat phyA3 promoter responsible for this regulation. Linker-scan mutagenesis of this promoter has identified two positive elements which together are necessary for maximal transcription in the absence of Pfr. These elements are designated PE1, centered at position -357 bp, and PE3, centered at position -96 bp. Sequence mutagenesis immediately downstream of PE3 results in maximal transcription in the presence of high Pfr levels, indicating that Pfr represses phyA3 transcription through a negatively acting sequence element. This element, designated RE1, with the sequence CATGGGCGCGG, encompasses a motif that is highly conserved in all monocot phyA promoters thus far characterized. DNase I protection analysis indicates that oat nuclear extracts contain multiple factors that bind to an array of sequence motifs, including PE1 and part of PE3, within 400 bp upstream of the oat phyA3 transcription start site. This DNA-binding pattern is not altered by Pfr. Weak binding to part of the RE1 motif is evident but also with no difference between high and low Pfr levels. We conclude that the signal transduction chain that mediates Pfr-directed repression of phyA3 transcription terminates with a negatively acting transcription factor that binds to the sequence element RE1.