The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal roles in inducing cell cycle arrest or in driving the cell towards apoptosis. Previously we had shown that, in a p53-dependent model of cell death, ceramide accumulated in a p53-dependent manner [Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA. J Clin Invest 1998;102:329-339]. In the current study, we investigated the biochemical pathways by which ceramide accumulated following p53 up-regulation. In both Molt-4 LXSN leukemia cells exposed to gamma-irradiation and in EB-1 colon cancer cells treated with ZnCl(2), p53 up-regulation led to de novo ceramide synthesis with predominance of N-palmitoylsphingosine (C16-ceramide) synthesis. The activation of the de novo pathway was not associated with increased activity of the key enzyme serine palmitoyltransferase (SPT) but rather with the increased activity of ceramide synthase. Furthermore, transcriptional up-regulation of the palmitoyl-specific Lass5 ceramide synthase gene was observed in Molt-4 but not in EB-1 cells. The SPT inhibitor ISP-1 or the ceramide synthase inhibitor fumonisin B1 led to substantial inhibition of ceramide accumulation in response to p53 up-regulation. Other biochemical pathways of ceramide generation such as sphingomyelinase activation were examined and found unlikely to contribute to p53-dependent ceramide formation. These studies indicate that p53 specifically drives de novo ceramide synthesis by activation of a ceramide synthase that favors the synthesis of N-palmitoylsphingosine.