Tumour necrosis factor (TNF)‐α induces a transient increase in N‐octanoylsphingosine (C8‐ceramide) which has been postulated as an intracellular mediator in TNF‐α signalling. We tested the ability of C8‐ceramide to reproduce the TNF‐α‐mediated interference with endothelial cell proliferation and DNA synthesis. TNF‐α (10 ng·mL⁻¹) and C8‐ceramide (20 µm) inhibited the incorporation of [³H]thymidine into DNA and led to an accumulation of cells in the G₁ phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF‐α and C8‐ceramide induced increased expression of p53. Treatment with TNF‐α or C8‐ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF‐α prevented RB phosphorylation whereas C8‐ceramide did not interfere with this process, suggesting that it follows a ceramide‐independent pathway.