The Crew Health System (CHeCS) plays a pivotal role in monitoring the life-support activities that maintain space station environmental quality and crew safety. Sampling hardware will be used in specific protocols to monitor the microbial dynamics of the closed spacecraft environment. NASA flight experience, ground-based studies, consultations with clinical and environmental microbiologists, and panel discussions with experts in engineering, flight-crew operations, microbiology, toxicology, and water quality systems all have been integral to the revision of in-flight microbial standards. The new standards for air and internal surfaces differentiate between bacterial and fungal loads, unlike previous standards that relied on total microbial counts. Microorganisms that must not be present in air or water or on surfaces also are listed. The proposed requirements and sampling protocols for air, surfaces, and water offer definitive standards and an effective monitoring strategy, and may mitigate the possibility of bioloads affecting crew health and spacecraft integrity.