Muropeptides contribute to the recognition of bacteria by modulating immune responses: the structural requirements for adjuvant activity were described in the seventies. During the last years, our knowledge of bacterial pattern recognition has increased dramatically and the importance of the absence of contaminations in both muropeptide preparations and other bacterial stimuli has become clear. We investigated a panel of 15 synthetic Limulus-negative muropeptides, four of them synthesized for the first time, as to their potency to synergize with lipopolysaccharide (LPS) in cytokine induction in human whole blood. No muropeptide was capable of stimulating cytokine release from human blood. However, as little as 20 nM of the muropeptides N-acetyl-muramyl-l-alanyl-d-isoglutamine (muramyl dipeptide, M(AdiQ)), N-acetyl-glucosamine-muramyl dipeptide GM(AdiQ), or C(18)M(AdiQ), which carries a non-natural additional fatty acid, sufficed to induce an up to 3 log-order shift in tumor necrosis factor alpha-release in response to 100 pg/ml LPS. The release of interleukin-1beta, interleukin-6, and interleukin-10 was also significantly enhanced although to a lesser extent. The synergistic effect was stereoselective with M(AdiQ) being the minimal active principle. Synergy was also observed on the transcriptional level by means of real-time PCR. Smaller molecules like N-acetylmuramic acid (M), aM, carrying a naturally occurring 1,6-anhydro-bound in M or M(A), containing only the amino acid l-alanine neither synergized with LPS nor influenced the synergy of other muropeptides with LPS. In conclusion, these data show that nanomolar quantities of muropeptides dramatically potentiate LPS-induced monocyte activation. This has implications for pyrogenicity testing and endotoxemia in patients.