The apoptosis-inducing factor was purified from the supernatant of PDBu-treated HL60 cells and recognized as human β₂m. The supernatant of PDBu-treated HL60 cells collected as in Materials and Methods was purified in a process using (A) the cation exchange FPLC (SP Sepharose HP; Pharmacia, NJ) and (B) the gel filtration HPLC (TSKgel G3000SW; Tosoh, Tokyo, Japan). The pool of active fraction eluted from gel filtration HPLC was applied to (C) the reverse-phase HPLC. The column (μBondasphere; Waters, MA) was eluted through a 90-minute linear time gradient from 28% to 40% acetonitrile. The flow rate was maintained at 2 mL/min. (D) The N-terminal sequence of the fraction with AIF activity. It is closely homologous with the N-terminal sequence of human β₂m. X shows an undetermined amino acid sequence. (E) The concentration of β₂m in the supernatant of PDBu-treated HL60 cells gradually increased over time for 72 hours.