Mitogen-activated protein kinases (MAPKs) have been implicated as regulators of differentiation. The biological effect of MAPK signaling in the nucleus is achieved by signal-responsive transcription factors. Here we have investigated MAPK signaling and activation of AP-1 transcription factors in P19 embryonal carcinoma cells undergoing cardiomyocyte differentiation. We show that aggregation and Me(2)SO treatment, which trigger the differentiation response, result in sustained activation of JNK1, p38, and ERK1/2 MAPKs and acquisition of AP-1 DNA binding activity. The induced AP-1 activity consists of c-Jun, JunD, and Fra-2 proteins and is accompanied with the increased expression of these proteins. JNK is involved in c-Jun phosphorylation, whereas ERK and p38 activities are essential for maximal c-Jun and Fra-2 expression, and AP-1 DNA binding activity. While the inhibition of ERK can partially prevent the formation of beating cardiomyocytes, the activity of p38 is absolutely required for the differentiation. Expression of dominant negative c-Jun(bZIP) in P19 cells can also inhibit the differentiation response. Surprisingly, however, expression of dominant negative SEK or JNK causes an inhibition of P19 cell proliferation. Taken together, the results show that ERK, JNK, p38, and AP-1 are activated in a coordinated and sustained manner, and contribute to proliferation and cardiomyocyte differentiation of P19 cells.