The role played by glutathione transferase P1-1 (GSTP1-1) in modulating the c-Jun N-terminal kinase (JNK) pathway has been extensively investigated using JNK isoforms known to exert opposite effects in the cells. We have expressed the isoform JNK1α2, that has been reported to transmit a pro-apoptotic signal, and we have analyzed both the phosphorylation level and the activity of this kinase in the presence of GSTP1-1. Contrary to what previous studies suggest, we found that GSTP1-1 is able to form a complex with the unphosphorylated and inactive JNK1α2 isoform, even in the absence of the substrate. We also analyzed the consequences of this interaction on the activity of both enzymes. The complex strongly reduced the activation of JNK1α2 while preserved GSTP1-1 from inactivation. Unexpectedly glutathione (GSH) exerted a negative effect on the affinity of GSTP1-1 for JNK1α2, suggesting that the intracellular levels of this thiol may allow a fine-tuning of the MAPK signaling pathway. Moreover we found that the adduct formed by GSH and the strong GSTP1-1 inhibitor NBDHEX abolishes the interaction between the GSTP1-1 and JNK1α2. These data confirm and extend at the molecular level previous evidence obtained in tumor cell lines.