Plasma viremia during HIV-1 infection is regulated by a dynamic balance between viral replication and removal of infected cells and cell-free virus. Administration of novel potent antiretroviral drugs provides an opportunity to study the consequences of perturbing this equilibrium by blockingde novoinfections. In this study, we examined the expression of differentially spliced forms of HIV-1 mRNA, unspliced (US) and multiply spliced (MS), in peripheral blood mononuclear cells (PBMCs) of patients treated with HIV protease inhibitors or combination therapy. In all nine patients studied, a significant reduction in the MS/US mRNA ratio was observed after 1 week of treatment, suggesting that the majority of HIV MS mRNA in the steady-state situation prior to therapy was expressed by cells which had been infected during the previous couple of days. This idea was supported by a detailed analysis of serial PBMC specimens collected from two of the patients during the first hours and days after initiation of therapy. In both cases, a substantial decrease in MS mRNA expression was evident already after 48 hr, whereas the expression of US mRNA at this time was virtually unaffected. These data indicate that the HIV mRNA splicing patternin vivois mainly determined by the relative proportion of newly infected cells and suggest that examination of this pattern could be useful in evaluating the potency of antiretroviral therapies and in studying dynamics of HIV-1 infection.