The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.