We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor- α (TNF-α) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30% inhibition) when challenged with TNF- α or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-α, its levels were measured at various times after the TNF-α preconditioning. We present evidence for the first time that, in normal brain cells, TNF-α pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-α. The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-α: 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B1, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.