Activation transcription factor-2 (ATF-2) is phosphorylated by various protein kinases, such as JNK/p38/ERK, calmodulin kinase IV, protein kinase A, and protein kinase C (PKC), in response to a variety of stimuli. However, the role of the phosphorylation of ATF-2 by PKC in vivo in the transcriptional control of genes that include the activation protein-1 (AP-1)/cyclic AMP-response element remains to be defined. Using antibodies against the phosphorylated serine residue (Ser(P)) at position 121 of ATF-2, we have demonstrated that PKC phosphorylates ATF-2 at Ser-121 and that phosphorylation of Ser-121 (to yield ATF-2pS121) becomes detectable at the late stage of the response of HeLa cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) and is maintained for more than 2 h. By contrast, phosphorylation of ATF-2 at threonine residues 69 and 71 (Thr-69/71, to yield ATF-2pT69/71) and at Ser-340 and Ser-367 (to yield ATF-2pS340 and ATF-2pS367) is detectable as an immediate early response. Unlike levels of ATF-2pT69/71 and ATF-2pS340, the level of ATF-2pS121 increases in the nuclei of HeLa cells in response to TPA. A serine-to-alanine mutation at position 121 of ATF-2 represses the c-Jun-dependent transcription of AP-1/cyclic AMP-response element reporter genes and also the p300-mediated activation of a Gal4-reporter gene in response to TPA. Our results suggest that the phosphorylation of ATF-2 at Ser-121 plays a key role in the c-Jun-mediated activation of transcription that occurs in response to TPA.