All experiments were performed in the absence of light. A 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) concentration of less than 10⁻⁴ M had no effect on cell growth of L cells. At higher concentrations, the cells were inhibited to levels which were similar to those obtained with equimolar doses of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). MIC inhibited the incorporation of ³H-thymidine by DNA more than that of ³H-uridine by RNA. Uptake of ³H from ³H-methyl-MIC by DNA was not influenced by the stage of the cell cycle. The greatest binding took place with DNA of the euchromatin fraction. MIC-treated DNA exhibited impaired template activity in vitro in the RNA polymerase s ystem but not with that of DNA polymerase. Chromatography of DNA hydrolysate from ³H-methyl-MIC-treated cells showed three major radioactive peaks, which corresponded to adenine, guanine and 7-methylguanine. Hydroxyurea markedly reduced the uptake of ³H by adenine and guanine but had relatively little effect on the ³H content of 7-methylguanine. Similarity of cytotoxic reactions of MIC to those of DIC supports the thesis that in the animal system DIC is metabolically converted to MIC, a potential methylating agent. Many of the effects of DIC can be accounted for by the action of MIC.