In human immunodeficiency virus type 1 (HIV-1) infection, the presence of divergent nucleotide sequences within a quasispecies has been associated with double infections or samples from different times or from different tissue compartments. The authors analysed HIV-1 proviral quasispecies from PBMC of three untreated Spanish patients displaying highly divergent nucleotide sequences without evidence of double infection. The origin of these nucleotide sequences was determined by phylogenetic analysis and by dating of the different groups using a genetic divergence versus sampling year plot from a set of Spanish samples. By their short genetic distance to the node of the patient's HIV-1 phylogenetic tree and by their early date of origin, close to the seroconversion time, some groups of sequences were considered ancestral. The presence within HIV-1 quasispecies of ancestral sequences, dated up to 10 years earlier than present ones, has important consequences for in vivo viral evolution, in the pathogenesis and treatment of HIV-1 infection.