Pyrrolidine dithiocarbamate (PDTC) is a stable anti-oxidant or pro-oxidant, depending on the situation, and it is widely used to inhibit the activation of NF-kappaB. We recently reported that PDTC activates the MIP-2 gene in a NF-kappaB-independent and c-Jun-dependent manner in macrophage cells. In this work, we found that PDTC activates signal transduction pathways in mouse ES cells. Among the three different mitogen-activated protein kinase (MAPK) pathways, including the extracellular-signal-regulated kinase (ERK), p38 MAP kinase, and stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathways, only the ERK pathway was significantly activated in mouse ES cells after stimulation with PDTC. Additionally, we observed a synergistic activation of ERK and induction of c-Fos after stimulation with PDTC in the presence of mouse embryonic fibroblast (MEF) conditioned medium. In contrast, another NF-kappaB inhibitor, BMS-345541, did not activate the MAP kinase pathways or induce expression of c-Fos. These results suggest that changes in the presence of the NF-kappaB inhibitor PDTC should be carefully considered when it used with mouse ES cells.