The multisite phosphorylation of the transcription factor ATF-2 was investigated using transformed embryonic fibroblasts from wild-type mice and mice deficient in c-Jun N-terminal kinases (JNK)1 and 2, and in the presence and absence of inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and the classical MAP kinase cascade. In wild-type cells, p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2 were not rate limiting for the phosphorylation of Thr69, Thr71 or Ser90. In JNK-deficient cells, p38 MAPK substituted for JNK partially in the phosphorylation of Thr69 and p38 MAPK or ERK1/2 in the phosphorylation of Thr71. JNK was the only MAP kinase that phosphorylated Ser90 under the conditions examined.