A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X-2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X-2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.