4(5)-(3,3-Dimethyl-1-triazeno)imidazole-5(4)-carboxamide (DIC) demonstrated alkylating activity when ¹⁴C-methyl DIC was administered to 2 patients po and to 2 rats ip. Fractionation of urinary purines from the first 6-hr urine samples obtained from these species showed a peak of radioactivity in the 7-methylguanine fraction, with only a trace of radioactivity in the adenine and guanine fractions. The 7-methylguanine was recrystallized to constant specific activity. Three rats were administered ¹⁴C-methyl DIC ip. After 5 hr the animals were sacrificed and the nucleic acids of the livers, kidneys, lungs, and brains were extracted, hydrolyzed, and subjected to ion-exchange chromatography. Radioactivity in the column effluent from the DNA and RNA of the livers was localized in the 7-methylguanine fraction with only a trace of radioactivity in guanine and adenine fractions. Hydrolyzates of nucleic acids obtained from the kidneys, lungs, and brains showed a significant radioactive 7-methylguanine peak, but the proportion of radioactivity in guanine and adenine was higher than that found in the liver nucleic acids. ¹⁴C-Methyl DIC was incubated with rat liver microsomes in vitro. Radioactive 7-methylguanine was isolated by ion-exchange chromatography of the ribosomal RNA hydrolyzate and from the supernatant fraction of the ribosomal reaction carried out in the presence of guanosine. Enzymatic conversion of DIC to 4(5)-(3-monomethyl-1-triazeno)imidazole-5(4)-carboxamide (MIC) which spontaneously generated a methylating intermediate, diazomethane, was proposed as a possible mechanism for the alkylating activity of DIC.