In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphoyctes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2^b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2K^b-restricted CTL specific for the OVA₂₅₈₂₇₆ peptide. This synthetic peptide and CNBr fragments of OVA (242285 and 242273) were able to target H-2^b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the K^b restricted determinant formed readily.