Human immunodeficiency virus type 1 (HIV-1) isolates recovered from infected children in Romania were characterized for their biologic, serologic, and molecular properties. The isolates were from subjects in different clinical states, and all showed cytopathic properties in peripheral blood mononuclear cells and varying kinetics of replication. The isolates grew to varying titers in macrophages and established T cell lines. Serologic evaluation with Romanian sera indicated stronger antibody response to the gp120 of Romanian isolates than to the envelope protein of HIV-1 isolates from other countries. Although there was cross-neutralization among the Romanian isolates, no substantial activity was noted against HIV-1 prototype strains from the United States, Africa, and Thailand. Genetic analysis of the envelope C2-V3 region strongly suggests that the Romanian isolates are a subtype distinct from those assigned to other HIV-1 strains analyzed to date. This finding raises questions about the origin of HIV-1 in Romania.