In October 2001, the greater New York City Metropolitan Area was the scene of a bioterrorism attack. The scale of the public response to this attack was not foreseen and threatened to overwhelm the Bioterrorism Response Laboratory’s (BTRL) ability to process and test environmental samples. In a joint effort with the Centers for Disease Control and Prevention and the cooperation of the Department of Defense, a massive effort was launched to maintain and sustain the laboratory response and return test results in a timely fashion. This effort was largely successful. The development and expansion of the facility are described, as are the special needs of a BTRL. The establishment of a Laboratory Bioterrorism Command Center and protocols for sample intake, processing, reporting, security, testing, staffing, and quality control are also described.