Aplidin™ is an antitumor drug that induces apoptosis and activates EGFR, Src, JNK and p38MAPK. Here, we show that Aplidin™ induces c-JUN,JUN B,JUN D, c-FOS,FRA-1 and FOS B genes of the activator-protein (AP)-1 family, and also p65/RELA, a major component of nuclear factor-kappa B (NF-κB). Concordantly, Aplidin™ increases AP-1 and NF-κB activity. c-FOS induction depends on EGFR, Src and JNK/p38MAPK. In contrast, induction of c-JUN does not require EGFR activity and p65/RELA induction is only partially dependent on these kinases. We used several genetically deficient cells to identify the critical target of Aplidin™. Mouse embryo fibroblasts (MEFs) deficient for src,yes and fyn, and those lacking all p38MAPK isoforms displayed normal Aplidin™ sensitivity (IC50=12 nM). In contrast, MEFs lacking jnk1 and jnk2, which do not express any JNK isoform, were much less sensitive (IC50>500 nM). Furthermore, cells lacking c-jun or expressing a c-Jun protein in which JNK targets Ser63/73 were mutated (c-JunAA) showed intermediate sensitivity (IC50=60 nM). Additionally, Aplidin™ has higher cytotoxic activity against proliferating than quiescent cells, which is reflected in higher JNK activation. We conclude that phosphorylation by JNK of c-Jun and additional substrate(s) is crucial for Aplidin™ activity.