Cell sorting of viable biological specimens has become widespread in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. This unit presents a revised and updated version of the biosafety guidelines for sorting of unfixed cells established in 1977 by the International Society of Analytical Cytology (ISAC), whose recommendations have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell-sorting experiments. The unit contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter setup, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment.