The third variable region (V3) of the envelope protein of human immunodeficiency virus type 1 (HIV-1) contains group- and type-specific epitopes for neutralizing antibodies and contains determinants involved in viral tropism and syncytium-inducing (SI) activity. We studied the in vivo relationship between V3 sequences and viral phenotypes in 24 perinatally HIV-1-infected children. To avoid in vitro selection of intrapatient minor variants, genetic studies were performed directly on uncultured peripheral blood mononuclear cells (PBMC), and the tropisms of HIV-1 isolates were evaluated by culturing patients' PBMC directly with monocyte-derived macrophages, lymphocytes, and MT-2 cells. According to their phenotypes, we could define five types of primary isolates: (i) non-syncytium-inducing (NSI) macrophagetropic, (ii) NSI macrophage-lymphotropic, (iii) NSI lymphotropic, (iv) SI lympho-T-cell line-tropic, and (v) SI pleiotropic. The SI viral phenotype was correlated with a more advanced status of disease. Genetic analysis of intrapatient molecular variants revealed that no relationship between the degree of intrapatient V3 variability and the pattern of viral tropism existed; moreover, within a single patient, the values for V3 variability between CD4+ lymphocytes and CD14+ monocytes were similar, thus suggesting that in vivo variability of the monocytotropic variants is more extensive than previously appreciated. A comparison between the intrapatient major variants and the phenotype of primary isolates disclosed that a negatively charged amino acid at residue site 25 was associated with an NSI macrophage- and macrophage-lymphotropic viral phenotype. Finally, by comparing the V3 sequences derived from our study population with those of several prototypes, we observed that the majority of isolates circulating in Italy are related to the North American subtype B macrophagetropic isolates.