Cell death occurs via two distinct mechanisms: necrosis and apoptosis. Apoptosis is an active suicidal response to various physiological or pathological stimuli, involved in embryonic development, tissue remodeling, and tumor regression, and the efficacy of anticancer agents depends on the target cells' propensity to undergo apoptosis. Thus, compounds inducing apoptosis in tumor cells are promising anticancer candidates. We explored new apoptosis inducers from microorganisms using human promyelocytic leukemia HL-60 cells as an indicator. An actinomycete strain RK95-74 (identified as Streptomyces sp., deposited as FERM P-15904) isolated from soil in Shiki City, Saitama Prefecture, Japan, produced a new potent apoptosis inducer, cytotrienin A, which contains triene ansamycin, 1-aminocyclopropane carboxylic acid, and 1-cyclohexene-1-carboxylic acid moieties. Cytotrienin A was obtained as a yellowish powder with mp 132~135°C and optical rotation [α]D5 +271° (c 1.0, MeOH), soluble in MeOH, CHCl3, and DMSO, and substantially insoluble in H2O and n-hexane. Its molecular formula was determined to be C37H48N2O8 by positive HR-EI-MS (Found: m/z 648.3355, Calcd: m/z 648.3407), supported by 1H and 13C NMR data. The UV spectrum (MeOH) showed absorption maxima at 262 nm (ε 45500), 273 nm (ε 46600), and 283 nm (ε 45400) (suggesting a triene moiety), and the IR spectrum (KBr) revealed absorptions at 3400, 1720, 1660, and 1000 cm-1. Cytotrienin A is closely related to mycotrienin II but differs in the 1-aminocyclopropane carboxylic acid moiety (found in plant metabolites but not in this ansamycin group). It showed antimicrobial activity against Pyricularia orizae with an MIC of 12.5 μg/ml but no activity against Staphylococcus aureus FDA 209P, Escherichia coli BE1186, Pseudomonas aeruginosa N-10 L-form, Candida albicans, or Chlorella vulgaris at 100 μg/ml. Cytotrienin A exhibited potent cytotoxicity against HL-60 cells (IC50 0.005 μg/ml) but low toxicity against human lung fibroblast WI-38 cells (>10 μg/ml). HL-60 cells treated with 0.01 μg/ml cytotrienin A for 16 hours showed condensed chromatin and fragmented nuclei (Hoechst Dye 33258 staining), and agarose gel electrophoresis of DNA revealed a ladder pattern (internucleosomal cleavage), indicating apoptosis. These data suggest that cell death induced by cytotrienin A is apoptosis. Further biological studies of cytotrienin A are in progress.