AbstractMice received inhalation exposures of 3H‐labeled ethylene oxide (EtO) gas at levels from 0.65 to 3.2 parts per million‐hours (ppm‐hr), which are below the exposure limits currently allowed for humans. Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a two‐week period and assayed for the amount of bound EtO. A strong increase in the level of EtO binding occurred in late spermatid stages; these stages are also genetically sensitive to the action of EtO. The maximum binding of EtO in late spermatids amounted to 6 × 103 alkylations/sperm head/ppm‐hr of exposure. Alkylation of the DNA within the sperm accounted for a very small fraction of the total sperm head alkylation, averaging about 20 DNA alkylations per sperm per ppm‐hr of exposure over the two‐week period. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm head alkylation and accounted for nearly all of the EtO binding. Protamine alkylation appears to be a significant cause of EtO‐induced genetic damage in spermiogenic cells of the mammal.