We show here that the anti-apoptosis protein Bcl-2 potently inhibits p53-dependent transcriptional activation of various p53-responsive promoters in reporter gene co-transfection assays in human embryonic kidney 293 and MCF7 cells, without affecting nuclear accumulation of p53 protein. In contrast, Bcl-2(Deltatransmembrane (TM)), which lacks a hydrophobic membrane-anchoring domain, had no effect on p53 activity. Similarly, in MCF7 cells stably expressing either Bcl-2 or Bcl-2(DeltaTM), nuclear levels of p53 protein were up-regulated upon treatment with the DNA-damaging agents doxorubicin and UV radiation, whereas p53-responsive promoter activity and expression of p21(CIP1/WAF1) were strongly reduced in MCF7-Bcl-2 cells but not in MCF7-Bcl-2(DeltaTM) or control MCF7 cells. The issue of membrane anchoring was further explored by testing the effects of Bcl-2 chimeric proteins that contained heterologous transmembrane domains from the mitochondrial protein ActA or the endoplasmic reticulum protein cytochrome b5. Both Bcl-2(ActA) and Bcl-2(Cytob5) suppressed p53-mediated transactivation of reporter gene plasmids with efficiencies comparable to wild-type Bcl-2. These results suggest that (a) Bcl-2 not only suppresses p53-mediated apoptosis but also interferes with the transcriptional activation of p53 target genes at least in some cell lines, and (b) membrane anchoring is required for this function of Bcl-2. We speculate that membrane-anchored Bcl-2 may sequester an unknown factor necessary for p53 transcriptional activity.