The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.