Metazoans express three unfolded protein response transducers (IRE1, PERK, and ATF6) ubiquitously to cope with endoplasmic reticulum (ER) stress. ATF6 is an ER membrane-bound transcription factor activated by ER stress-induced proteolysis and has been duplicated in mammals. Here, we generated ATF6 alpha-and ATF6 beta-knockout mice, which developed normally, and then found that their double knockout caused embryonic lethality. Analysis of mouse embryonic fibroblasts (MEFs) deficient in ATF6 alpha or ATF6 beta revealed that ATF6 alpha is solely responsible for transcriptional induction of ER chaperones and that ATF6 alpha heterodimerizes with XBP1 for the induction of ER-associated degradation components. ATF6 alpha(-/-) MEFs are sensitive to ER stress. Unaltered responses observed in ATF6 beta(-/-) MEFs indicate that ATF6 beta is not a negative regulator of ATF6 alpha. These results demonstrate that ATF6 alpha functions as a critical regulator of ER quality control proteins in mammalian cells, in marked contrast to worm and fly cells in which IRE1 is responsible.