Monocyte—macrophages are important target cells and reservoirs for HIV. The existing methods for the quantification of infectious virus in HIV stocks are not totally satisfactory for use with macrophage cultures. We have developed an infectious focus assay for the direct quantification of virions infectious for human peripheral blood monocyte-derived macrophages adhering to plastic microtitre plates. The combination of an HIV-1 p24-antigen-specific monoclonal antibody and a β-galactosidase-linked second antibody resulted in a sensitive and very specific assay. With 5-bromo-4-chloro-3∼indolyl-β-D-galactopyranoside as substrate, the assay proved to be as sensitive as p24 antigen quantification in culture supernatants.