The human immunodeficiency virus (HIV) is characterized by extremely high variability resulting in the emergence of widely divergent viral strains in diverse geographical locations and different populations. HIV strains can also vary significantly with regard to their biological and immunological properties, which may have important implications for clinical aspects of HIV infection, diagnostics, treatment, and the development of effective HIV vaccines. It is therefore important to develop appropriate laboratory technologies and capacities for systematic collection and detailed characterization of globally prevalent HIV-1 strains. This second edition contains the latest information and recommendations with regard to standard procedures for HIV isolation and its genetic, biological, and immunological characterization with a special emphasis on their applicability in HIV vaccine-related research. The laboratory methods described in these guidelines were intensively validated through various collaborative studies conducted in the framework of the WHO-UNAIDS Network for HIV Isolation and Characterization.