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To study viral heterogeneity at a very early phase of primary HIV-1 infection.Samples were drawn very early during primary HIV-1 infection. A virus population-based approach was used to study the viral heterogeneity in the C2–V3 and p17 regions.Plasma samples (n = 33) were obtained before or shortly after onset of acute symptoms in 15 patients. In all subjects, the first sample was drawn within 10 days after onset of symptoms. Peripheral blood mononuclear cells (PBMC) were available in two patients. The number of polymorphic sites in the C2–V3 (15 patients) and p17 regions (eight patients) were determined by direct sequencing.The sequence heterogeneity was restricted in most patients, although only two out of 15 patients had a completely homogeneous C2–V3 sequence. However, pronounced individual differences were seen. Rapid sequence changes occurred during the first month in two patients. In one patient, the major DNA species at day 12 later became the major species in plasma.The viral population is seldom completely homogeneous during primary HIV-1 infection, although the heterogeneity is restricted in most, but not all, patients. These individual differences do not seem to be due to sex or viral subtype. Rapid changes of the virus population may occur during primary HIV-1 infection. The DNA species detected in PBMC do not only represent earlier viral quasispecies but are also a potential source of future viral RNA species.