The mechanism of action of 2,2'-(methylenediimino)bis-1,3,4-thiadiazole (NSC 143019) was clarified by studies on its effects on monolayer cultures of growing cells of the mouse cell line BALB/3T3. At concentrations below 50 muM, NSC 143019 specifically inhibited DNA and RNA syntheses without appreciably affecting protein synthesis. The syntheses of DNA and RNA were inhibited equally and concomitantly by the compound. The inhibition was reversed by removal of the compound and was prevented competitively by an equimolar amount of nicotinamide. It was also reversed completely by guanosine (0.1 mM) or deoxyguanosine (0.1 mM) and was reversed partially by xanthosine (1 mM). Other nucleosides did not influence the inhibition. The inhibition of DNA synthesis by NSC 143019 was not due to inhibition of RNA synthesis, and vice-versa. NSC 143019 inhibited the conversion of [8-14C]hypoxanthine to acid-soluble and -insoluble guanine nucleotides but not to adenine nucleotides. It was strongly suggested from these results that at concentrations of NSC 143019 below 50 muM the primary action of this compound might be due to the inhibition of GMP biosynthesis at the step of conversion of IMP to xanthosine 5'-phosphate.