The question which detailed structures of bacterial modulins determine their relative biological activity and respective host cell receptors was examined with synthetic variants of mycoplasmal lipopeptides as model compounds, as well as recombinant outer surface protein A (OspA) of Borrelia burgdorferi and lipoteichoic acid. Mouse fibroblasts bearing genetic deletions of various toll-like receptors (TLR) were the indicator cells to study receptor requirements, primary macrophages served to measure dose response. The following results were obtained: (i) the TLR system discriminates between modulins with three and those with two long-chain fatty acids in their lipid moiety, in that lipopeptides with three fatty acids were recognized by TLR2, whereas those with two long-chain fatty acids and lipoteichoic acid required the additional cooperation with TLR6; (ii) substitution of the free N terminus of mycoplasmal lipopeptides with an acetyl or palmitoyl group decreased the specific activity; (iii) removal of one or both ester-bound fatty acids lowered the specific activity by five orders of magnitude or deleted biological activity; (iv) oxidation of the thioether group lowered the specific activity by at least four orders of magnitude. The implications of these findings for physiological inactivation of lipopeptides and host-bacteria interactions in general are discussed.