Transforming growth factors (TGFs) reversibly induce the anchorage-independent growth of nontransformed cells. TGF activity is often monitored by the growth of normal rat kidney (NRK) fibroblasts in soft agar, and it is known that more than one growth factor is involved in the regulation of their soft agar growth. To more clearly define the growth factors responsible for the soft agar growth of NRK cells, the effects of four growth factors were examined: platelet-derived growth factor (PDGF); TGF-beta; epidermal growth factor (EGF); and fibroblast growth factor (FGF). This study determined that PDGF induces the soft agar growth of NRK cells, in both plasma-supplemented medium and serum-free medium supplemented with FGF, and neither TGF-beta nor an EGF-related growth factor is required for this effect. It was also determined that FGF, which alone does not induce the soft agar growth of these cells, potentiates the responses of NRK cells to various combinations of PDGF, TGF-beta, and EGF. Interestingly, the effect of TGF-beta was found to depend on the growth factor composition of the medium. In the absence of EGF, TGF-beta partially inhibits the soft agar growth response of NRK cells to PDGF, whereas, in the presence of EGF, TGF-beta increases their response to PDGF. These findings indicate that at least four unrelated growth factors regulate the anchorage-independent growth of NRK cells. These findings have important implications for the use of NRK cells to assay TGFs.