Nurses are the largest group of health-care professionals, yet they are not uniformly educated regarding transplantation and organ donation. The future of transplantation hinges on education of this group. Before meaningful studies can be conducted, an instrument to measure attitudes and commitment to organ transplantation is needed. The purpose of this study was to examine content and construct validity as well as establish internal reliability of an investigator-developed online instrument to measure nurses’ attitudes and commitment to organ transplantation by registered nurses. The online instrument was administered to registered nurses enrolled in transplantation electives at the University of Alabama in Huntsville and Vanderbilt University. Exploratory factor analysis revealed 4 components with eigenvalues over 1.0. The components were as follows: (1) desire to work in transplantation, (2) confidence in transplantation advocacy, (3) organ donation advocacy, and (4) procurement. Internal consistency of the revised instrument was established (α = .94). The Transplant-Registered Nurse (TXP-RN) instrument is a new instrument with excellent reliability and validity that can be used to measure attitudes and knowledge of American nurses about organ donation and transplantation. This important step is necessary before educational interventions can be accurately assessed.