The use of allogeneic human bone, dura, and fascia has achieved an enduring and accelerating role in the augmentation of spinal fusions and the repair of skeletal and dural defects. Primary sterilization of these nonviable cadaveric tissues magnifies the potential sources and ensures the microbiological sterility of the implant. Subsequent lyophilization facilitates preservation and distribution and reduces the immunogenicity of the graft. The evaluation of gaseous ethylene oxide (EO) as a sterilant was suggested by the delerious effects of alternative methods. Through a series of experiments, the following properties of EO sterilization were studied: (a) surface and interstitial sterilization; (b) the diffusion of EO into tissue, the formation of the reaction products ethylene chlorohydrin (EC) and ethylene glycol (EG), and the desorption of all three from tissues; (c) lyophilization and aeration in the removal of residues; and (d) minimization of residues through pretreatment. Gaseous EO is a very effective surface sterilant of wet bone, dura, and fascia and does not grossly alter these tissues. Its partial penetration through compact bone renders it less reliable for an interstitial antimicrobial effect, unless access to the interior is provided by serial openings. The toxicity of EO, EC, and EG mandates the desorption through lyophilization of these compounds (EC and EG are formed during sterilization with EO). Before sterilization, bone must be rid of marrow by vigorous irrigation with deionized water. The resultant reduction of the number of cells and of the available chloride decreases antigenicity and the formation of EC. Freeze-drying for more than 72 hours, in some cases augmented by prolonged aeration at room temperature, reduces EO, EC, and EG to acceptable levels. The accurate assay of residues in tissue requires acetone extraction for gas chromatography on rehydrated tissues because extraction of dry tissues gives falsely low results. Rigorous adherence to a protocol incorporating these findings justifies the acceptance of gaseous EO as a safe, relatively rapid, and inexpensive sterilant of bone and soft tissues.