A primary isolate (KMT) of human immunodeficiency virus type 1 (HIV-1) resistant to recombinant soluble CD4 (rsCD4) was isolated from an HIV-1-infected individual and grown in a T lymphoid cell line. KMT isolate passaged on CEM cells (KMT/CEM) was still resistant to rsCD4. The V1/V2 and V3 regions of the viral envelope glycoprotein are thought to be involved in various biological phenotypes. To determine the exact envelope region of the KMT isolate responsible for sensitivity to rsCD4 and cellular tropism, we performed sequence analysis of KMT and KMT/CEM isolates. Sequence analysis of the KMT isolate showed that the sequence of the V3 region was relatively homogeneous, whereas a considerable heterogeneity of the V1/V2 region was noted. In contrast, the sequences of the V1 to V3 regions were homogeneous in KMT/CEM isolates. Analysis of NL4-3-based recombinant viruses with amplified sequences of the V1 to V3 regions from KMT and KMT/CEM isolates showed that the V1/V2 region modulated the sensitivity to rsCD4. A change in resistance to rsCD4 by the V1/V2 region was associated with the ability of the isolate to replicate in macrophages and efficiently replicate in T lymphoid cell lines. A change to an isolate sensitive to rsCD4 was associated with reduced replication efficiency in T lymphoid cell lines. Our results suggest that the V1/V2 region is involved in modulating the sensitivity to rsCD4, macrophage tropism, and replication efficiency in T lymphoid cell lines.