Background: Virus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage. Results: The heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found. Conclusion: This study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance. © 2009 Rozera et al; licensee BioMed Central Ltd.