We studied whether cell detachment from the matrix, observed during ceramide-induced apoptosis, is secondary to completion of the apoptotic program. CHP-100 neuroepithelioma cells exposed to N-hexanoylsphingosine (C(6)-Cer) underwent detachment from the substrate and apoptosis with slow kinetics. Apoptotic cells were fairly completely recovered in the detached fraction, that, differently from the adherent counterpart, displayed the hallmarks of caspase 3 activation, as well as poly-(ADP)ribose polymerase (PARP) cleavage and focal adhesion kinase (FAK) downregulation. A key role for caspase 3 in apoptosis execution was suggested by the evidence that its selective inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited cell death. However, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (targeting not only caspase 3 but also caspases 1, 5, 7, 8 and 9) did not prevent ceramide-induced cell detachment, although apoptosis, caspase 3 processing, PARP cleavage and FAK downregulation were suppressed in floating cells. These results demonstrate that ceramide-induced cell detachment is upstream activation of effector caspases. We discuss the possibility that ceramide-induced cell detachment might be instrumental to apoptosis execution.