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To compare the dynamics of HIV-1 molecular markers in peripheral blood mononuclear cells (PBMCs) and in plasma during the asymptomatic phase of untreated HIV-1 infection.Using seminested real-time PCR assays, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the PBMCs of 10 untreated HIV-1-infected men at multiple time points during the asymptomatic phase of infection and compared the longitudinal trends of these markers with those of viral RNA in plasma.Whereas plasma RNA levels did not significantly change in any of the individuals, levels of usRNA significantly increased with time in six out of 10 persons, and levels of prDNA in four. Slopes, changes, and time-weighted changes from baseline of usRNA, prDNA, and CD4+ cell count, but not of plasma RNA, were significantly different from zero (P < 0.01). No significant longitudinal trend of plasma RNA was observed in the study group using linear mixed models, whereas the trends of usRNA, prDNA, and CD4+ cell count were highly significant (P < 0.001). usRNA levels increased significantly faster than those of plasma RNA or prDNA, suggesting a temporal increase in viral replication rates in PBMCs. Finally, CD4+ cell count inversely correlated with levels of usRNA and prDNA, but not with plasma RNA level.During the asymptomatic phase of untreated HIV-1 infection, when virion production and clearance are balanced, resulting in stable plasma viremia, viral load in PBMCs steadily increases and is a sensitive and direct longitudinal virological marker of infection progression.