Immunization of C57BL/6 (B6) mice with heat-killed Sendai virus generates a Sendai virus-specific CD8+ T cell response. This suggests that APC have the capacity to take up and present exogenous (nonreplicative) Sendai virus Ag on MHC class I molecules. Little is known about the intracellular requirements for processing of this form of Ag and its presentation on MHC class I. Therefore, we have studied the processing and presentation of heat-killed Sendai virus Ag on MHC class I molecules in splenic APC. Heat-killed Sendai virus Ags were efficiently processed by normal B6 as well as by TAP-1(-/-) splenic APC. Presentation was MHC class I restricted, since no presentation was seen by APC from TAP-1/beta2m-/- mice that lack expression of MHC class I. Presentation occurred even in the presence of brefeldin A, but was blocked by cytochalasin D as well as chloroquine. Finally, B6 as well as TAP-1(-/-) splenic APC, loaded with heat-killed Sendai virus Ag in vitro, primed naive CD8+ T cells in vivo. These studies suggest the existence of a TAP-independent pathway for Ag presentation on MHC class I in normal splenic APC, bearing many similarities with the MHC class II pathway for Ag presentation. The present results are discussed in relation to the events underlying the processing and presentation of exogenous Ag on MHC class I, the molecular basis for CD8+ T cell priming during viral infections, and prospects for vaccine development.