In response to the Ebola outbreak in 2014, many hospitals designated specific areas to care for patients with Ebola and other highly infectious diseases. The safe handling of category A infectious substances is a unique challenge in this environment. One solution is on-site waste treatment with a steam sterilizer or autoclave. The Johns Hopkins Hospital (JHH) installed two pass-through autoclaves in its biocontainment unit (BCU). The JHH BCU and The Johns Hopkins biosafety level 3 (BSL-3) clinical microbiology laboratory designed and validated waste-handling protocols with simulated patient trash to ensure adequate sterilization. The results of the validation process revealed that autoclave factory default settings are potentially ineffective for certain types of medical waste and highlighted the critical role of waste packaging in successful sterilization. The lessons learned from the JHH validation process can inform the design of waste management protocols to ensure effective treatment of highly infectious medical waste.KEYWORDS Ebola, sterilization, medical waste, serious communicable diseases, autoclave T he Ebola outbreak in West Africa in 2014 revealed potential gaps in the abilities of U.S. hospitals to safely provide care for patients with highly infectious diseases. Prior to the outbreak, the capacity to care for patients in the United States infected with high-consequence pathogens was limited to a few specialized facilities, or biocontainment units (BCUs) (1-3). In response to the crisis, the Centers for Disease Control and Prevention (CDC) recommended a tiered approach wherein U.S. hospitals serve as frontline health care facilities, Ebola assessment hospitals, or Ebola treatment centers (ETCs) (4). The Office of the Assistant Secretary for Preparedness and Response (ASPR), a federal office in the Department of Health and Human Services (HHS), created a regional response plan, which called for the creation of Regional Ebola and Other Special Pathogen Treatment Centers (RETCs) (5). These RETCs were modeled in part on the U.S. facilities that provided care for Ebola patients, namely the University of Nebraska Medical Center, the National Institutes of Health, Emory University, and Bellevue Hospital Center, and also include design elements based on local capabilities and lessons learned from the outbreak (6).