Within solid tumor microenvironments, lactic acidosis, and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here, we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Furthermore, these data suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells. Cancer Res; 72(2); 491–502. ©2011 AACR.