The phenotypic features of liver sinusoidal endothelial cells (SEC), open fenestrae in sieve plates and lack of a basement membrane, are lost with capillarization. The current study examines localization of CD31 as a marker for the dedifferentiated, nonfenestrated SEC and examines regulation of SEC phenotype in vitro. CD31 localization in SEC was examined by confocal microscopy and immunogold-scanning electron microscopy. SEC cultured for 1 day express CD31 in the cytoplasm, whereas after 3 days, CD31 is also expressed on cell-cell junctions. Immunogold-scanning electron microscopy confirmed the absence of CD31 surface expression on fenestrated SEC 1 day after isolation and demonstrated the appearance of CD31 surface expression on SEC that had lost fenestration after 3 days in culture. SEC isolated from fibrotic liver do show increased expression of CD31 on the cell surface. Coculture with either hepatocytes or stellate cells prevents CD31 surface expression, and this effect does not require heterotypic contact. The paracrine effect of hepatocytes or stellate cells on SEC phenotype is abolished with anti-VEGF antibody and is reproduced by addition of VEGF to SEC cultured alone. VEGF stimulates SEC production of nitric oxide. NG-nitro-L-arginine methyl ester blocked the paracrine effect of hepatocytes or stellate cells on SEC phenotype and blocked the ability of VEGF to preserve the phenotype of SEC cultured alone. In conclusion, surface expression of CD31 is a marker of a dedifferentiated, nonfenestrated SEC. The VEGF-mediated paracrine effect of hepatocytes or stellate cells on maintenance of SEC phenotype requires autocrine production of nitric oxide by SEC.