Human immunodeficiency virus type 1 strain 89.6 is a dualtropic isolate that replicates in macrophages and transformed T cells, and its envelope mediates CD4-dependent fusion and entry with CCR5, CXCR-4, and CCR3. To map determinants of cofactor utilization by 89.6 and determine the relationship between cofactor use and tropism, we analyzed recombinants generated between 89.6 and T-cell-tropic (HXB) or macrophage-tropic (JRFL) strains. These chimeras showed that regions of 89.6 env outside V3 through V5 determine CXCR-4 utilization and T-cell line tropism as well as CCR5 utilization and macrophage tropism. However, the 89.6 env V3 domain also conferred on HXB the ability to use CCR5 for fusion and entry but not the ability to establish productive macrophage infection. CCR3 use was conferred on HXB by 89.6 env V3 or V3 through V5 sequences. While replacement of the 89.6 V3 through V5 region with HXB sequences abrogated CCR3 utilization, replacement of V3 or V4 through V5 separately did not. Thus, CCR3 use is determined by sequences within V3 through V5 and most likely can be conferred by either the V3 or the V4 through V5 domains. These results indicate that cofactor utilization and tropism in this dualtropic isolate are determined by complex interactions among multiple env segments, that distinct regions of the Env glycoprotein may be important for utilization of different chemokine receptors, and that determinants in addition to cofactor usage participate in postentry stages in the virus replication cycle that contribute to target cell tropism.