Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.