Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response. © 2004 Elsevier Inc. All rights reserved.