The stimulation of cells with physiological concentrations of insulin induces a variety of responses, like an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71-phosphorylation precedes the induction of ATF2-Thr69+71-phosphorylation by several minutes. We previously found that in JNK-/- fibroblasts cooperation of the ERK1/2 and p38-pathways is required for two-step ATF2-Thr69+71-phosphorylation in response to growth factors. As JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, next to ERK1/2 and p38 in the insulin-induced two-step ATF2-phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71-phosphorylation was sensitive to MEK1/2-inhibition with U0126 and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK-inhibitor SP600125 or expression of dominant negative JNK-activator SEK1 prevented the induction of ATF2-Thr69+71-, but not ATF2-Thr71-phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38-activation with SB203580 or expression of dominant negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71-phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2-preceded JNK-translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK rather than p38 underlies the two-step insulin-induced ATF2-phosphorylation in JNK-expressing cells.