We previously reported the development of a src-specific tumor regression system in chickens in which preinfection with rASV1702, a mutant of Rous sarcoma virus (RSV) encoding non-myristylated src product with a novel N-terminal domain, results in the immune suppression of challenge tumors induced by RSV. In order to adapt this system to the mouse, we have developed NIH3T3 and Balb/c3T3 (B3T3) cell lines that express 1702src, v-src, c-src, and other src variants, either by transfection or by infection with packaged recombinant Moloney virus (MLV) vectors. The sequence of the 1702 src cDNA, produced by reverse transcription-polymerase chain reaction (RT-PCR), confirmed the previously suggested 1702src N-terminal domain structure, fusing six amino acids from Pr76gag and 39 amino acids of env signal peptide sequence to Ala-76 of src. These cell lines were characterized for src expression and activity, cell compartmentalization of src product, tyrosine phosphorylation substrate specificity and transforming and tumorigenic potential. Based on these parameters, murine cell lines expressing 1702src were characteristically similar to chicken embryo fibroblasts (CEFs) infected with rASV1702. Finally, B3T3/1702src expressors, or membrane fractions of these cells, induced src-specific tumor protection in syngeneic mice against v-src-transformed tumor challenges. Splenic lymphocytes isolated from Balb/c mice inoculated with B3T3/1702src cells showed in vitro cytotoxicity against B3T3/v-src cells but not against untransformed B3T3 cells. Antibodies specific for Lyt2, mouse CD3 and H-2Dd blocked this cytotoxicity, whereas those specific for L3T4 did not, suggesting MHC class I-restricted, CD8-mediated cell killing. These data indicate that Balb/c3T3-expressed 1702src induces a cellular anti-tumor immunity based on src-specific tumor rejection antigens.