Apoptosis is an essential mechanism of cell death required for normal development and homeostasis of all multicellular organisms. Smac/DIABLO is a dimeric protein important in the control of apoptosis by removing the inhibitory activity of IAPs (inhibitor of apoptosis proteins). In vitro studies reveal that dimerization is required for its function. Here we investigate the structural and thermodynamic features of folding and dimerization of Smac/DIABLO. To disturb the folded, dimeric structure, we used high hydrostatic pressure, low and high temperatures, and chemical denaturing agents. Conformational changes were monitored using spectroscopic techniques such as fluorescence and circular dichroism (CD) as well as gel filtration chromatography. Our data show that Smac/DIABLO is very stable under pressures up to 3.1 kbar, even at subzero temperatures. A complete denaturation/dissociation process is obtained when we use high concentrations of urea, which affect its secondary structure as assessed by CD. The association of pressure and subdenaturing urea concentrations also results in complete denaturation/dissociation of the protein. Under these conditions, unfolding of the protein shows concentration dependence that is in accordance with the dimer-monomer dissociation equilibrium, confirming Smac/DIABLO dissociation. These results suggest that most of the treatments lead to a reversible disruption of the dimeric structure with a dissociation constant ( K d) of 34 x 10 (-21) M (34 zM). This tight dimer is biologically relevant, considering that monomeric mutants bind IAP with low affinity. The extremely high stability of the dimeric form of Smac/DIABLO also implies that once expressed in the cell the protein has a low probability of dissociation and, consequently, loss of function. In addition, the stability in the zeptomolar range is the highest so far measured for a dimeric protein. It also indicates that under most circumstances Smac/DIABLO does not exist as a monomer in the cell and suggests that the dimer-to-monomer equilibrium does not play a regulatory role in the Smac/DIABLO-IAP interaction.