Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) is a new member of the TNF superfamily. In this work, the key role of Zn(2+) in high-level expression of soluble TRAIL was confirmed. The yield of soluble TRAIL reached 1.6 g l(-1) using a novel, two-stage Zn(2+) feeding strategy, and the accumulation of TRAIL inclusion bodies decreased. Furthermore, the purified TRAIL showed stronger cytotoxicity activity against human pancreatic 1990 tumor cells as the molar ratio of Zn(2+) to TRAIL monomer was 2 in purified TRAIL solution.