Protein phosphorylation is commonly used to modulate transcription factor activity. However, all existing genetic evidence for stimulation of transcription factor activity by phosphorylation rests on loss-of-function mutations. To demonstrate conclusively that phosphorylation of a transcription factor potentiates its transactivation potential in vivo, we constructed a c-Jun mutant that is phosphorylated by the cAMP-sensitive protein kinase A (PKA) instead of the UV- and Ras-responsive protein kinase JNK. The transcriptional activity of this mutant is enhanced by PKA, but not by JNK activation. These results provide a positive and conclusive proof that phosphorylation of c-Jun on a critical site (Ser73) located in its activation domain is directly responsible for enhancing its transactivation function.