Litmomycin, an antibiotic isolated from Streptomyces litmogenes, is highly active against Gram-positive bacteria and possesses antitumor activity. It inhibited viral DNA polymerase activity in vitro. The amount of litmomycin required to cause 50% inhibition of enzyme activity was 80-100 mug (180-225 nmoles)/ml of reaction mixture. The enzyme inhibition was observed when polyriboadenylate--oligodeoxythymidylate, polydeoxyadenylate-oligodeoxythymidyate, polyribocytidylate-oligodeoxyguanylate, activated DNA, and 70 S RNA were used as templates. Reaction kinetics and the mechanism of enzyme inhibition are discussed. The results suggest that litmomycin interacts with the template primer and not with the enzyme protein to stop the polymerization process.