Genetic and epigenetic alterations affecting proteins involved in apoptosis can contribute to the establishment and progression of cancer. Recently, our laboratory has isolated a novel gene, TMS1, that is aberrantly methylated and silenced in a significant proportion of human breast cancers. TMS1 contains a caspase recruitment domain (CARD), suggesting a role in caspase-mediated cell death. In the present study, we characterize the participation of TMS1 in apoptosis and examine the subcellular localization of the protein. Inducible expression of TMS1 inhibited cellular proliferation and induced DNA fragmentation in a time-dependent manner. These apoptotic events were blocked by the general caspase inhibitor, Z-VAD-fmk. The ability of TMS1 to trigger apoptosis was also suppressed by a dominant negative form of caspase-9 but not by a dominant negative form of caspase-8, indicating that TMS1 functions through activation of caspase-9. Unlike a number of other CARD-containing proteins, TMS1 did not activate nuclear factor kappaB-dependent transcription, consistent with a proapoptotic role for TMS1 in death signaling pathways. Timed localization studies revealed that TMS1-induced apoptosis was accompanied by the redistribution of TMS1 from the cytoplasm to perinuclear spherical structures. Whereas the apoptotic activity of TMS1 was blocked by caspase inhibition, the formation of TMS1-containing subcellular structures was not, suggesting that the redistribution of TMS1 precedes caspase activation. Both the proapoptotic activity of TMS1 and aggregate formation were dependent on the CARD. In summary, the data indicate that TMS1-induced apoptosis proceeds through a CARD-dependent aggregation step followed by activation of a caspase-9-mediated pathway.