Recent studies indicate that T cell cross-priming preferentially occurs against long-lived, stable proteins. We have studied cross-priming by using the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), a protein that normally is not MHC class I cross-presented. This study shows that a C-terminally truncated, noncleavable variant of LCMV-GP led to the accumulation of stable, soluble GP trimers in the endoplasmic reticulum (ER) of the antigen donor cell, and thereby converted LCMV-GP into a potent immunogen for cytotoxic T lymphocyte cross-priming. Immunization of mice with tumor cells expressing an ER-retained LCMV-GP variant cross-primed protective antiviral cytotoxic T lymphocyte responses in vivo at least 10,000-fold better than immunization with cells expressing the cross-presentation-"resistant" wild-type LCMV-GP. Thus the ER is a cellular compartment that can provide antigen for cross-presentation, and modifications affecting stability and subcellular localization of the antigen significantly increase its availability for MHC class I cross-presentation. These findings impinge on vaccine strategies.