Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.