Virus-host cell interaction plays a decisive role on viral biology. The Orthopoxvirus Vaccinia virus (VV) is a large double-stranded DNA virus (≈200 Kpb) that replicates exclusively in the cytoplasm of infected host cells. Our group have previously shown that VV activates the mitogen-activated protein kinases (MAPKs) ERK1/2 (Andrade et al., 2004) and JNK (PEREIRA, manuscript in preparation), which are both required for an efficient VV multiplication. Moreover, we have shown that the transcription factor c-Jun, which is a common signal transducer of the above pathways, is activated from 3 up to 48 h.p.i. (hour post-infection). Interestingly, there is some c-Jun "temporal-regulation", since MEK/ERK1/2 activates c-Jun until 24 h.p.i. and MKK4/JNK activates it after 24 h.p.i (PEREIRA, manuscript in preparation). Based on these observations, we decided to investigate the biological significance of c-Jun to VV. Thus, we generate cell lines stably-expressing c-Jun dominant-negative mutation. Clones expressing dominant-negative (DN) mutation, i.e. those where Jun expression upon VV infection was drastically reduced, were then used for infectivity assays. Then, A31 cells or the clones selected were serum-deprived (1% FSB) for 12h and then infected at multiplicity of infection (MOI) 10 or with 100-200 PFU. Cell lysates were then prepared to: single-step virus yield, western blot or dot blot. We had observed a reduction in virus yields in the DN cells as compared to that expressing wild-type c-Jun, the parental cell line A31. The time observed in this experiment, 24 h.p.i., allow us to associate this decrease with the MEK/ERK1/2 pathway. We have also observed a reduction in Egr-1 induction after infection with VV in the DN cells, which is also required for an efficient VV multiplication (SILVA, manuscript in preparation). Taken together, these data suggest that the signaling pathway involving MEK/ERK1/2/c-Jun/Egr-1 is necessary for an efficient viral production. In addition, we observed a remarkable decrease in VV plaque size and a delay in the replication of viral DNA in the DN cells. Altogether, our results demonstrated that the transcription factor c-Jun is activated during the VV infection as an attempt to generate the most adequate intracellular environment to its own multiplication.