We isolated a subtype B infectious DNA clone of human immunodeficiency virus type 1 (HIV-1) from a seronegative patient with acute infection and determined the entire nucleotide sequence. All the reading frames encoding the structural proteins (Gag, Pol, and Env) and nonstructural proteins (Tat, Rev, Vpr, Vif, and Nef) were found. Although most functional domains in these proteins were conserved, we identified a duplication of the T cell factor 1alpha (TCF-1alpha) element in the long terminal repeat and many variations in the N-linked glycosylation sites in the V4-V5 region but not in the V1-V3 loop of Env, compared with prototype subtype B clones. Furthermore, phylogenetic analysis of the entire nucleotide sequence indicated that this HIV-1 was distinct from the prototype subtype B clones, suggesting that transmitted viruses can be variants. This HIV-1 DNA clone will be a useful prototype for investigating the mechanism of HIV-1 transmission.