The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.