The mode of action of tomaymycin (TM) which is a new antibiotic with antitumor activity was studied. Inhibition of the incorporation of labeled precursors into the acid-insoluble fraction of Bacillus subtilis cells indicated that TM inhibits nucleic acids biosynthesis, while having relatively little effect on protein synthesis. TM displaced methyl green (MG) from a MG-DNA complex and a TM-DNA complex was isolated by gel filtration chromatography. The formation of the TM-DNA complex resulted in (1) shifts of UV absorption profiles of TM and the corresponding difference spectra, (2) increase in the melting temperature of DNA, (3) protection of native DNA from degradation by nuclease O. These findings are compatible with the proposition that TM exerts its action as a growth inhibitor by forming a complex with DNA which in turn prevents the DNA from participating as a template in the biosynthesis of nucleic acids.