Four neonates who were infected with a single unit of blood from a human immunodeficiency virus-1 (HlV-l)-infected adult (patient 1) were studied. Two of the infected children (patients II and HI) developed symptomatic HIV-1 disease and died within the first 3 y of life. One child (patient IV) died at 8 mo of age of clinical problems that may have been HIV-related. In contrast, one child (patient V) has remained asymptomatic for 7.5 y and has exhibited a very gradual decline in CD4+ cell number. A previous study had shown very limited sequence diversity of isolates from patients I, II, and III (McNearney T et al.: Proc Natl Acad Sci USA 87:1917-1921, 1990). The current study examined additional HIV-1 sequences encoding the principal neutralizing V3 loop of the surface envelope protein of isolates from patients I and V. Amplified sequences were obtained using the polymerase chain reaction from a cultured isolate and uncultured peripheral blood leukocytes, and nucleotide sequences were determined for 13 clones from patient I and 19 clones from patient V. Clones derived from the cultured isolate exhibited less predicted amino acid sequence diversity on average (0-5.2%) than did sequences from uncultured leukocytes (0-19.8% differences). All clones were more closely related to those from patients II and III (0-19.8% amino acid differences) than to other North American or European isolates (18.8-27.0% amino acid differences) or African isolates (41.0-48.0% amino acid differences). Substitutions occurred at sites predicted to modulate host cell tropism and proteolytic cleavage of the V3 loop. Late time-point isolates from uncultivated leukocytes diverged by 6.319.8% from earlier time-point isolates or from those isolates derived from cultured leukocytes. Half of the amino acid positions at which differences occurred between earlier and late time-point HIV-1 strains were shared in isolates from patients I and V. This demonstrates that nonrandom mutations occur in the evolution of HIV-1 genotypes during natural infection.