Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase-8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN-amplified NB, our results indicate that the transcriptional inactivation of caspase-8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon-gamma (IFN-gamma) induce caspase-8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN-amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN-amplified and MYCN-single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5' terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase-8. The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans-acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.