Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA‐restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer‐related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA‐A2‐bound peptides from HLA‐A2⁺ ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA‐A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8⁺ CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA‐A2‐restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA‐A2⁺ tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide‐pulsed targets. Furthermore, lack of recognition of several HLA‐A2⁺ control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL‐defined antigens that could lead to the development of multi‐epitope vaccines for the treatment of ovarian cancer.