The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2 alpha) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2 alpha in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2 alpha. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2 alpha phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2 alpha phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2 alpha phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eIF2 alpha. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2 alpha phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.