S-nitroso-N-acetyl-D,L-acetylpenicillamine (SNAP), a chemical donor of NO, inhibited serum- and basic fibroblast growth factor (bFGF)-stimulated cultured endothelial cell (EC) proliferation in a dose-dependent manner. The inhibitory effect of NO was reversible after washoff of SNAP-containing media. Measurement of nitrate and nitrite in the media of SNAP-treated EC indicated that decomposition of SNAP into NO reached a stable level at or before 24 h; proliferation of EC was significantly inhibited for another 48 h and recovered thereafter if no additional SNAP was added. The level of NO produced by inhibitory concentrations of SNAP was comparable to NO levels produced by the induction of inducible nitric oxide synthase (iNOS) in smooth muscle cells or retinal pigmented epithelial cells. The growth-inhibitory effect of NO was unlikely to be due to cytotoxicity since 1) cells never completely lost their proliferative capacity even after 10 days of exposure to repeated additions of SNAP, 2) the inhibitory effect was reversible upon removal of NO and with the passage of time, and 3) NO did not reduce the number of cells that were growth-arrested with TGF-beta 1. In addition to its mitogenic effect, bFGF induced pronounced phenotypic changes, including suppression of contact inhibition, altered cell morphology, and scattering of the cells, in BPAEC cultures, whereas cells treated simultaneously with bFGF and NO did not exhibit these changes. These observations suggest that NO contributes to the regulation of angiogenesis and reendothelialization, processes that require EC proliferation, migration, and differentiation.