The monocyte activation test (MAT) has been introduced as an alternative for the detection of pyrogens in pharmaceuticals with the rabbit pyrogen test or the Limulus amebocyte lysate (LAL) test. The basis of the MAT is that pyrogens, via Toll-like receptors (TLRs) expressed on monocytes, stimulate cytokine production. Here, we report that, at concentrations that did not induce whole blood cytokine production when tested separately, (1-->3)-beta-D-glucans powerfully co-stimulated cytokine production (IL-6/IL-8) induced by ligands for TLR1/2, TLR2/6, TLR4, and TLR5. Experiments were performed to investigate the involvement of particular (1-->3)-beta-D-glucan receptors such as dectin-1. Spleen tyrosine kinase (Syk) inhibition attenuated the potentiating effects of (1-->3)-beta-D-glucans on TLR-induced cytokine production, suggesting that dectin-1 was involved. However, experiments with low molecular (1-->3)-beta-D-glucans such as laminarin argued against the involvement of dectin-1 in the co-stimulatory effects of (1-->3)-beta-D-glucans. Thus, although the receptors involved in the co-stimulatory actions of (1-->3)-beta-D-glucans on TLR-induced cytokine production are yet to be elucidated, it is clear that (1-->3)-beta-D-glucans may greatly affect MAT results and, when undetected in pharmaceuticals, may give rise to serious side-effects in patients co-exposed to other elicitors of innate immunity, such as during infections.