The ability of seven methylating agents to form 7-methylguanine and O sup 6-methyl-guanine was compared to their ability to initiate carcinogenesis as measured by the initiation of GGTase-positive foci. The seven methylating agents studied were diazald, DMH, DMN, DMS, MMS, MNNG, and MNU. The DNA methylation and initiation of GGTase-positive foci was determined in partial hepatectomized rats. The formation of foci was promoted by 500 ppm sodium phenobarbital in the drinking water. While six of the seven compounds (DMH, DMN, DMS, MMS, MNNG and MNU) produced 7-methylquanine, only the four compounds (DMH, DMN, MNNG and MNU) that produced O sup 6-methylguanine initiated GGTase-positive foci. The extent of O sup 6-methylguanine produced by the methylating agents did not correspond with their potency to initiate GGTase-positive foci. Therefore, the initiation of GGTase-positive foci required the formation of O sup 6-methylguanine. However, some sequential event altered the quantitative relationship of O sup 6-methylguanine formation to the incidence of GGTase-positive foci.