Lipoproteins activate cells of the innate immune system via heteromers of Toll-like receptor (TLR) 2 with either TLR1 or TLR6. In spite of progress in understanding TLR-dependent signal transduction and the pathophysiological relevance of TLR2, the molecular basis of ligand recognition by this receptor is poorly defined. Here, we show that the bioactivity of lipopeptides (LP) critically depends on the dilution protocol and especially the presence of proteins or detergents acting as solubilizers. Fluorescence correlation spectroscopy of fluorescently labeled analogs of synthetic LP revealed that the LP form aggregates in solution. Dilution into protein- and serum-free buffers led to a complete loss of activity due to formation of large and highly heterogeneous aggregates. When dimethylsulfoxide stock solutions were diluted into BSA or serum-containing buffers particles of strongly reduced size were obtained. For some LP, an intermediary dilution step either with tert.-butyl alcohol/H2O (4:1) or with octyl-beta-D-glucopyranoside further increased activity. For a panel of LP exhibiting very different activities when diluted directly into protein-containing solutions, introduction of this dilution step resulted in comparable bioactivities. These results demonstrate the significance of solubilizing agents for the bioactivity of LP and are highly relevant for analyzing structure-activity relationships of LP-dependent TLR2 activation.