The c-Myc transcription factor is involved in the regulation of cellular proliferation and differentiation and is one of the most frequently deregulated genes in human cancers. While c-Myc is known to enhance the proliferative potential of cells, its activation in immortalized fibroblasts has been found to result in apoptosis following gamma-irradiation or under adverse growth conditions, including serum deprivation and hypoxia. When plating Rat-1 fibroblasts at low cell densities (100 cells/100 mm plate), we observed a substantial reduction in the clonogenicity of cells with deregulated c-Myc activity compared to cells with normal c-Myc activity. This difference in clonogenicity was apparent despite the fact that cells were plated in media containing sufficient serum and oxygen concentrations known to suppress apoptosis of exponentially growing Rat-1 fibroblasts with activated c-Myc. Therefore, we hypothesized that the observed reduction in plating efficiency in cells with activated c-Myc occurred via an apoptotic mechanism and that a fibroblast-derived factor was required for suppression of apoptosis. Overexpression of the anti-apoptotic oncogene, Bcl-2, in cells with activated c-Myc restored the plating efficiency to normal levels in cells plated at low cell densities. This strongly suggested that the decreased clonogenicity of fibroblasts with altered c-Myc activity resulted from enhanced apoptosis of the cells under these conditions. Furthermore, plating cells on a feeder layer of lethally-irradiated fibroblasts or in Rat-1 conditioned media increased the plating efficiencies of sparsely plated cells in a dose-dependent fashion. These results suggest that in addition to previously reported requirements for serum-derived growth factors and normal oxygen conditions, a paracrine factor liberated by Rat-1 fibroblasts is required to suppress c-Myc-induced apoptosis in these cells.