HAMLET (human α-lactalbumin made lethal to tumor cells) is a complex of human α-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α-lactalbumin is sufficient to induce cell death. We used the bovine α-lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine α-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of α-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of α-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.