Paracrine interaction between preadipocytes and microvascular endothelial cells may play a role in the regulation of adipose tissue growth. We report here a study of the effect of extracellular matrix factors secreted by microvascular endothelial cells, derived from adipose tissue, on preadipocyte differentiation in primary culture. Extracellular matrix components (EC) were prepared by differential centrifugation of medium conditioned by microvascular endothelial cells (CM). Preadipocyte differentiation was assessed by enumerating cells containing Oil-Red-O-stainable neutral lipids and by assaying cellular triacylglycerol (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) specific activity. Both supernatant (containing soluble components) and pelleted (containing large complexes of EC) fractions of CM stimulated preadipocyte differentiation. When the supernatant fraction was used, the proportion of cells containing visible lipid droplets was 29% ± 3% of total preadipocytes in the presence of extracellular complexes, as compared with 6% ± 1% under control conditions. This differentiation induction was associated with fourfold increases in TG content and GPDH specific activity. Neither the supernatant nor the pelleted fraction of EC affected the maximal differentiation induced by hormonal stimulation in serum-supplemented or serum-free media. The major EC, fibronectin, laminin, and collagen IV, had no effect on differentiation when added individually to culture medium. Collection of CM under hyperglycemic (18 mmol/L glucose) compared with control (6 mmol/L glucose) conditions reduced the stimulatory effect of extracellular complexes by twofold, suggesting decreased or altered production by endothelial cells. The present findings demonstrate that microvascular endothelial cells release EC that promote preadipocyte differentiation. Previously, we have shown that microvascular endothelial cells also release factor(s) that stimulate preadipocyte replication in vitro. In vivo, both proliferative and differentiating factors may act together to promote the expansion of adipose tissue. Thus, microvascular endothelial cells may play a significant role in the development and growth of adipose tissue.