The surfaces of three chitosan samples, differing only in their degrees of deacetylation and of carboxyethyl chitosan were chemically characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and Fourier transform infrared, both before and after sterilization with ethylene oxide. Unexpected elemental ratios suggest that surface chemical modification occurred during the processing of the original chitin, with further surface modification on subsequent sterilization, despite previous reports to the contrary. Cell viability was evaluated by direct contact methyl thiazole tetrazolium and lactate dehydrogenase assays between the chitosan particles and A549 human epithelial cells, which demonstrated that the modifications incurred on sterilization are reflected in biocompatibility changes. All the samples were found to be biocompatible and nontoxic before sterilization and remained so subsequently. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.