Two new WRKY transcription factors from parsley (Petroselinum crispum), WRKY4 and WRKY5, were isolated using the yeast one-hybrid system. In yeast, both proteins interacted sequence-specifically with W boxes (TTGACC) and activated transcription. They appear to contain functional leucine zippers, which increase their affinities for W boxes. Co-transfection experiments in parsley protoplasts confirmed their in vivo-binding specificity for W boxes. Elicitor-mediated expression of the WRKY5 gene, the first parsley member of the group III family of WRKY proteins, is extremely transient, with high mRNA levels occurring within a time window of less than 1 h. WRKY4 and -5, as well as the previously identified parsley transcription factors WRKY1 and -3, are encoded by immediate early elicitor-activated genes that differ in their sensitivity to cycloheximide (CHX) and their activation kinetics. We propose that a number of the pathways activated during the plant defense response require the induction of several distinct WRKY transcription factors with different DNA binding-site preferences to fine-tune the activation of a wide spectrum of target genes.