Cultured vascular endothelial cells (ECs) secrete transforming growth factor beta (TGF-β) which stimulates intimal smooth muscle cells to synthesize increased amounts of lipoprotein-binding proteoglycans. We report here that the amount of TGF-β1 synthesized by ECs is correlated with EC density and cell spreading. ECs cultured at low density synthesized and secreted 2- to 3-fold more TGF-β, measured by the Mink lung cell assay, than intermediate and high density cultures, and this increase in secreted protein was matched by a corresponding increase in mRNA for TGF-β1 measured by Northern hybridization using a [32P]-labeled cDNA probe for TGF-β1. TGF-β, mRNA was detected in individual cells by in situ hybridization with the cDNA probe labeled with [35S] and with a [35S]-labeled 30-mer antisense oligonucleotide. In situ mRNA levels did not relate to cell density per se but to cell area. The larger the area of substratum covered, the more mRNA per cell, irrespective of cell density. For cell areas in the range 500–1,200 µm2, a doubling of cell area resulted in an approximately 3-fold increase in mRNA. Cells cultured at low density had a larger mean cell area than cells cultured at higher densities, and this increased area was sufficient to account for the increased TGF-β1 mRNA levels found for the low density cultures by Northern hybridization. Despite variations in cell area, cell volumes did not change significantly with cell density. These findings may have implications for the development of atherosclerotic lesions, since the area of endothelial cells is known to increase over plaques, in regions of altered shear stresses and also with age.