The macrocyclic polyketide kendomycin exhibits antiosteoporotic and antibacterial activity, as well as strong cytotoxicity against multiple human tumor cell lines. Despite the promise of this compound in several therapeutic areas, the cellular target(s) of kendomycin have not been identified to date. We have used a number of approaches, including microscopy, proteomics, and bioinformatics, to investigate the mode of action of kendomycin in mammalian cell cultures. In response to kendomycin treatment, human U-937 tumor cells exhibit depolarization of the mitochondrial membrane, caspase 3 activation, and DNA laddering, consistent with induction of the intrinsic apoptotic pathway. To elucidate possible apoptotic triggers, DIGE and MALDI-TOF were used to identify proteins that are differently regulated in U-937 cells relative to controls. Statistical analysis of the proteomics data by the new web-based application GeneTrail highlighted several significant changes in protein expression, most notably among proteasomal regulatory subunits. Overall, the profile of altered expression closely matches that observed with other tumor cell lines in response to proteasome inhibition. Direct assay in vitro further shows that kendomycin inhibits the chymotrypsin-like activity of the rabbit reticulocyte proteasome, with comparable efficacy to the established inhibitor MG-132. We have also demonstrated that ubiquitinylated proteins accumulate in kendomycin-treated U-937 cells, while vacuolization of the endoplasmic reticulum and mitochondrial swelling are induced in a second cell line derived from kangaroo rat epithelial (PtK(2)) cells, phenotypes classically associated with inhibition of the proteasome. This study therefore provides evidence that kendomycin mediates its cytotoxic effects, at least in part, through proteasome inhibition.