Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Δlgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Δlgt-LTA by human peripheral blood cells. In this study, we demonstrate that Δlgt-LTA–induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Δlgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA–mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Δlgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Δlgt-LTA–mediated immune activation and appear to induce the phagocytic uptake of Δlgt-LTA via engagement of FcγRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.