Studies show that n-3 polyunsaturated fatty acids (PUFA) inhibit proliferation and induce apoptosis in cancer cells. Recent reports indicate that this effect is due to activation of the unfolded protein response (UPR). However, what causes this activation has been unclear. We examined the effects of eicosapentaenoic acid (EPA) on the human leukemia cell line HL60 and the econazole (Ec) resistant HL60 clone E2R2. Ec depletes Ca(2+) from the ER and blocks Ca(2+) influx in mammalian cells, leading to activation of the UPR and apoptosis. EPA inhibited growth of HL60 cells strongly, while E2R2 cells were much less affected. Gene expression analysis of HL60 cells revealed extensive changes in transcripts related to the ER homeostasis, Ca(2+)-homeostasis and cell cycle/apoptosis. Protein levels of phosphorylated eIF2alpha, a selective translation inhibitor and UPR hallmark, activating transcription factor 4 (ATF4) and sequestosome-1 were moderately increased, whereas the cell cycle/progression protein cyclin D1 was decreased in HL60. In contrast, EPA concentrations that strongly inhibited and caused activation of the UPR in HL60 cells had no effect on the expression level of these UPR markers in E2R2 cells. Given that the only known difference between these cells is Ec-resistance, our results strongly suggest that the inhibitory effect of EPA on HL60 cells is initially meditated through alterations of the Ca(2+)-homeostasis followed by activation of the UPR.