Many mouse and human tumours express major histocompatibility complex (MHC) class I-associated antigens that constitute targets for syngeneic cytotoxic T lymphocytes (CTL). Genes encoding such antigens were isolated from a mouse mastocytoma and from human melanomas by genetic methods. Isolation and characterization of MHC class I-associated peptides has enabled specific anchor residues to be identified that are typical of peptides that bind to distinct class I molecules. Moreover, CTL specific to particular MHC-peptide combinations have been used to identify naturally occurring antigenic peptides in cell extracts and enabled them to be sequenced directly. Most known MHC ligands are of viral origin or are self peptides derived from normal proteins. Here we use total acid extraction and repeated fractionation to isolate and sequence Lewis lung carcinoma (3LL)-specific peptide(s), which shows sequence homology to the connexin 37 protein. Synthetic octamers based on these sequences bind to 'empty' H-2Kb molecules on RMA-S cells, sensitize RMA-S cells to lysis by specific anti-3LL CTL, and induce anti-tumour CTL. The tumour-associated peptide originates from mutated connexin 37 expressed in 3LL.