Mononuclear phagocytes play an important role in human immunodeficiency virus (HIV) infection. However, knowledge of the extent of macrophage involvement in the pathogenesis of HIV infection is still incomplete. This is due in part to the technical difficulties associated with isolating, purifying, and culturing these cells. Isolation of a pure population of human monocytes is essential when studying the role of these cells in HIV infection. In addition, the differentiation, activation, and replication of human monocytes are critically affected by both isolation and culture conditions. This review examines the different monocyte isolation and purification methods and culture conditions that are currently being employed and assesses their suitability for studying the functions of monocytes in HIV infection.