Avian defective sarcoma virus mutant LA46 carries a temperature-sensitive defect in replication and transformation. To elucidate this defect, we cloned the integrated provirus of LA46. By DNA-mediated transfection, the cloned DNA induced fusiform-transformed foci in chick embryo fibroblasts without helper virus. LA46-encoded transformation-specific protein p105 was expressed in these transformants in the absence of helper virus-encoded proteins. Superinfection of the transformed cells with different helper viruses resulted in the rescue of pseudotypes. All the rescued pseudotypes retained the temperature-sensitive phenotype in virus replication and transformation, suggesting that the defect was due to a cis-acting lesion in the LA46 genome. Restriction enzyme comparison between LA46 and wild-type virus revealed sequence differences near the 5' and 3' termini of the LA46 genome, including the long terminal repeat regions.