Growth of human diploid [embryo lung T16-1] fibroblasts on the cell sheets treated with various fixatives and enzymes was examined to investigate the mechanism of the density-dependent inhibition of cell proliferation. Growth was inhibited about 40% when fibroblasts were cultured on glutaraldehyde-fixed cell sheets of late passage cells, compared with cell growth on perchloric acid-fixed cell sheets or in control cultures without cell sheets. Treatment of glutaraldehyde-fixed cell sheets with heparitinase or nitrous acid caused the complete loss of the inhibitory effect. When fibroblasts were cultured on glutaraldehyde-fixed cell sheets derived from early middle and late passage cell cultures, cell growth on the late passage cell sheets showed greater inhibition. These inhibitory effects of the cell sheets correlated well with both the relative amount of heparan sulfate to the total glycosaminoglycans in the cell layer and to the saturation density at each passage. Apparently heparan sulfate (or its complex) on the cell surface is involved in the density-dependent inhibition of cell proliferation.