Incorporation of [³H]-thymidine into DNA in non-synchronized cultures of human endothelial cells was blocked by a 24 h exposure to TNF in a dose dependent manner that resulted in accumulation of cells in G1, as assayed by flow cytometry analysis of DNA content. Proliferation restarted when cells were replated in the absence of TNF. Northern analysis of c-myc mRNA in synchronized untreated cultures showed a transient increase previous to DNA synthesis that was decreased with TNF treatment. Western analysis of the retinoblastoma gene product RB in untreated synchronized cultures showed reduced electrophoretic mobility during the transition from G1 to S, congruent with RB inactivation by phosphorylation. TNF treatment prevented RB retardation and reduced total levels of RB protein. Taken together our results show that the TNF-mediated block of endothelial proliferation correlates with deficient activation of the G1 events necessary for entry into S, despite the presence of serum and endothelial mitogens.