The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca(2+) and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.