The tumor suppressor gene p53 is mutated or deleted in more than 50% of human tumor cells, resulting in down-regulation of p53-inducible genes such as the CDK inhibitor p21WAF1. Natural products that activate the p21 promoter and upregulate its expression are potential cancer chemotherapeutics. In a screening program for p21 promoter activators, we found three actinomycete strains producing compounds that enhance luciferase activity in H1299/tsp53-Luc cells, all of which produce trichostatin derivatives. One strain, RK98-A74 (isolated from Okutama, Tokyo soil and identified as Streptomyces sp.), produced a new trichostatin derivative, trichostatin RK (TSRK). TSRK was purified via acetone extraction, ethyl acetate partition, silica gel chromatography, and preparative HPLC. Structural analysis using UV, FAB-MS, 1H/13C NMR, COSY, HMQC, and HMBC revealed that TSRK differs from trichostatin A (TSA) by a methyl group substituting the hydroxyl group in the hydroxamic acid moiety of TSA. Activity assays showed TSA induced over 4-fold luciferase activity at 50 ng/ml, while TSRK, trichostatin C, monomethyl TSA, and dimethyl TSA had negligible activity. The hydroxamic acid moiety of TSA is critical for high-affinity binding to the zinc ion in the active site of histone deacetylase (HDAC), and methyl substitution in TSRK obstructs this binding, explaining its low activity.