The clinical significance of persistent residual viremia in patients on prolonged highly active antiretroviral therapy (HAART) is not clear. Moreover, it remains to be demonstrated whether residual viremia consists of viruses capable of spreading infection in vivo upon termination of therapy. Using residual viral RNAs (vRNAs) isolated from a HAART-treated patient's plasma, we cloned full-length viral genomes and found that most of them could produce infectious, replication-competent HIVs when transfected into TZM-bl cells, suggesting that residual viruses produced in the absence of therapy can initiate fresh cycles of infection and spread in host cells. The data further indicate that residual viremia may pose a major concern with regard to the emergence of drug-resistant HIVs during periods of low adherence to therapy.