The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. 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 c-Jun N-terminal kinase (JNK)−/− fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, 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 MAPK kinase (MEK) 1/2-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 SAPK kinase (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.