SUMMARYA number of factors of possible significance in elucidating the mechanism of inactivation of spores exposed to the alkylating agent, ethylene oxide (ETO), were investigated in this study. The sterilant system consisted of a mixture of ethylene oxide (12%) end dichlorodifluoro‐methane (88%). Spores were exposed to ETO end suspended in distilled water overnight to determine if the ETO treatment would effect release of protein, RNA, DNA, or DPA. Only DPA release was effected, it being appreciably greeter from exposed than unexposed spores. Pretreatment of spores with agents postulated to effect the intactness of the spore coat was noted to alter ETO resistance characteristics. The resulting survivor curve exhibited en unusually long death lag period followed by an increased first‐order death rate. Such an alteration, however, could be counteracted by increasing the time between pretreatment end ETO exposure. A comparison of lyophilized vegetative cells, germinated spores, and heat‐activated spores revealed that the ETO resistance of germinated spores was closer to that of heat‐activated spores than to that of vegetative cells. To obtain a logarithmic death rate for heat‐activated spores, it was necessary to precondition at about 98% R.H. prior to the normal preconditioning et 33% R.H. Vegetative cells end germinated spores, however, did not require preconditioning other then et 33% R.H. The effect of increasing time of ETO exposure on germination end postgerminative end vegetative development of spores in a rich medium end in a deficient medium was evaluated. No significant change in rate or efficiency of germination was observed for either medium. However, the postgerminative phase of development of treated spores was notably lengthened over that of untreated spores in either medium. Treated spores which were incubated in the rich medium entered the vegetative phase of growth before those incubated in the deficient medium, this difference becoming more pronounced with increasing periods of exposure.