The relationship between the genetic variability in the V3 loop and the biological characteristics of 38 biological clones from 5 European and 7 African HIV-1 isolates belonging to 3 different subtypes (subtype A, B, and D) was investigated. Seventeen of 19 clones displaying a syncytium-inducing (SI) capacity had a positively charged amino acid located at position 11 and/or 25 in the V3 loop sequence. All 19 non-syncytium-inducing (NSI) virus clones lacked such a positive charge at the same positions (p < 0.001). The mean of net charge in the overall V3 loop sequences of the SI clones was higher than that of the NSI clones (p < 0.001). Within the same strains, the SI clones replicated faster/higher than the NSI clones in peripheral blood mononuclear cells (p < 0.01), but not in CD4+ T cell cultures (p > 0.1). All SI clones but only 5 of 19 NSI clones could replicate in human continuous T cell and monocytic cell lines (p < 0.001). A higher number of positively charged amino acid substitutions was found among the subtype D SI clones. Only one of eight autologous sera tested had the ability to neutralize the contemporaneously isolated NSI clones, but not the SI clones. This study indicates that the V3 loop amino acid sequences of HIV-1 biological clones from different origins belonging to different genetic subtypes are clearly correlated with viral syncytium-inducing capacity, cell tropism, and replication rate.