We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.