Mammary fat may affect human breast cancer development. In this study a novel coculture system was used to investigate stromal-epithelial cell interactions in vitro. The extent of differentiation of a feeder layer of 3T3-L1 preadipocytes affected the clonal growth of breast cancer cells under anchorage-independent conditions. When the feeder-layer preadipocytes were proliferating, the clonal growth of estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 and MDA-MB-436 cells was stimulated by 100%, 43%, and 60%, respectively. In contrast, after differentiation the feeder layer of mature adipocytes inhibited clonal growth of breast cancer cell lines by 60%. No clonal growth stimulation of human pancreatic (Panc-1) or prostatic (PC-3) cancer cells was noted when these cells were grown under identical conditions with a proliferating 3T3-L1 cell feeder layer. These findings suggest that proliferating preadipocytes may be associated with factors that specifically stimulate breast cancer cell growth.