Macrophages express both CXCR4 and CCR5 coreceptors, but restrict X4 HIV-1 replication unless the Env-V3 region, a major determinant of cell tropism, is exchanged with that of R5 HIV-1. As the V3 exchange concomitantly alters the nucleotide sequences, we introduced silent mutations in the V3 or C2 region of macrophage-tropic R5 JRFL without changing the amino acids. Immunoblot analysis confirmed that viral proteins including Env-gp120 were similarly incorporated in wild-type (wt) and mutant virions. The silent mutants infected CCR5-positive MAGIC5 cells but not CCR5-negative MAGI cells, as productively as wt viruses, indicating that the silent mutations did not alter coreceptor utilization. In contrast, two of three silent V3-mutant viruses failed to replicate efficiently in primary macrophages, whereas other V3- or C2-mutants and wt JRFL infected macrophages productively. Furthermore, synthesis of the full-length viral DNA of the aberrant V3-mutant was largely reduced in macrophages. These results suggest that V3 nucleotide sequences may be one of the postentry factors restricting HIV-1 replication in macrophages.