The integrins are heterodimeric receptors that bind proteins in the extracellular matrix (ECM), activate intracellular signaling pathways and regulate programmed cell death. Disruption of cell-ECM interactions in epithelial cells results in the onset of programmed cell death, also called cell detachment-induced apoptosis or anoikis. The intracellular lipid second messenger ceramide appears to play a role in apoptosis. Here we report our studies of cell detachment-induced apoptosis in chicken embryo (CE) cells. Cells removed from tissue culture dishes by trypsinization, were either maintained in suspension or plated on fibronectin-coated dishes under serum free conditions. Cells maintained in suspension started undergoing apoptosis 8 hours after plating and the proportion of apoptotic cells in suspension, compared to cells plated on fibronectin, continued to increase upto 24 hours. The ceramide content of cells maintained in suspension relative to cells plated on fibronectin increased 4 hours after plating the cells and continued to increase upto 24 hours. Thus, the increase in ceramide levels in the absence of an integrin signal preceded the onset of apoptosis. Primary mouse embryo fibroblasts maintained in suspension also underwent cell detachment-induced apoptosis and contained elevated levels of ceramide compared to cells attached to fibronectin. CE cells treated with sodium orthovanadate, the phorbol ester PMA, or transformed with activated c-Src(Src-Y527F) were protected from apoptosis and contained lower ceramide levels compared with control cells. Exposure of cells plated on fibronectin to exogenous sphingomyelinase resulted in a marked accumulation of ceramide and subsequent apoptosis of the treated cells. The induction of apoptosis in sphingomyelinase treated cells was similar to that detected in cells maintained in suspension. These data suggest that the absence of an integrin signal is correlated with an elevation of ceramide levels and that increasing the ceramide content in attached cells is sufficient to trigger apoptosis within the treated cells.