We found that transcription from the promoter in the long terminal repeat of Rous sarcoma virus in rat 3Y1 cells is dependent on the presence of serum in the culture. However, this serum dependence of transcription was relieved when 3Y1 cells were transformed by the oncogene v-src. Crossfeeding experiments showed no evidence for the production of a serum-substituting extracellular growth factor by the transformed cells. Using 3Y1 cells transformed with temperature-sensitive Rous sarcoma virus, we showed that the tyrosine kinase activity of pp60v-src was responsible for the serum-sparing effect on the level of RNA expressed from the viral promoter. Sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases that nonspecifically elevates the level of phosphotyrosine-containing proteins in cells, stimulated transcription from the viral promoter. The effects of both pp60v-src and orthovanadate were resistant to cycloheximide. These results suggest that the serum independence of transcription from the viral promoter in v-src-transformed cells was probably due to the constitutive activation of intracellular growth-factor pathways by the tyrosine kinase activity of pp60v-src.