Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [ 14C]dT/[ 3H]dC ratios for whole sperm and sperm DNA were the same for each animal tested. The spermatozoa of males used in the dosimetry studies were labeled with [ 14C]thymidine, and then the animals were given various [ 3H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of 3H and 14C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide ( E/ N) ranged from ∼ 10 −7 at 3.3 mg/kg up to ∼ 10 −4 at 400 mg/kg, and the data indicated that E/ N increased with the 1.5 power of the dose. E/ N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA. The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.