Adrenocortical carcinoma (ACC) is a highly malignant tumor with poor response to classical antitumor therapy. Steroidogenic acute regulatory (StAR) protein is expressed in most human ACCs. The aim of this study was to induce antitumoral T cells directed against StAR in a murine tumor model. Because a suitable syngenic adrenocortical mouse tumor model is lacking, we established a clone of the mouse myeloma Sp2-0 tumor cell line stably expressing murine StAR (Sp2-mStAR). Using repeated im injections of plasmid DNA encoding mStAR followed by infection with a recombinant vaccinia virus (rVV) expressing mStAR, we induced a cytotoxic T-cell response as measured by enzyme-linked immunospot assay. To demonstrate antitumor activity of the vaccination procedure, mice were treated as follows: group A, mice immunized with plasmids and rVV encoding mStAR receiving Sp2-mStAR cells; control group B, mice immunized with the empty plasmid and the empty rVV receiving Sp2-mStAR cells; control group C, mice immunized with the empty plasmid and rVV encoding P450 side-chain cleavage enzyme receiving Sp2-mStAR cells; and control group D, mice immunized with plasmid and rVV encoding mStAR receiving parental Sp2-0 cells. A high proportion (89-100%) of the control groups B, C, and D developed subcutaneous tumors. In contrast, immunization specific for mStAR (group A) was highly protective against tumor growth (percentage of tumor-free animals, 67%; P < 0.001 vs. controls). In summary, these results show that T-cell tolerance toward mStAR can be broken, resulting in antitumoral immunity. Thus, StAR represents a candidate target antigen for immunotherapeutic strategies against ACC.