Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis.Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels.A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner.Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.