Abstract β-glucans are pharmacologic agents that rapidly enhance host resistance to a variety of biologic insults through mechanisms involving macrophage activation. To determine whether stimulation of the β-glucan receptors on human monocytes resulted in cytokine production, monolayers of monocytes were incubated with purified yeast glucan particles and measured for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein. By Northern blot analysis, TNF-α mRNA was detected within 30 min of incubation with glucan particles, peaked at 2 h, and remained elevated for at least 8 h. Glucan induction of IL-1β mRNA followed a similar time-course of initiation and accumulation. By enzyme-linked immunosorbent assays (ELISAs), significant levels of TNF-α and IL-1β were present in supernatants of glucan-treated cells within 1 h and plateau levels of both cytokines were approached within 4 h. At particle-to-cell ratios of from 0.4 to 18, glucan particles induced dose-dependent increases in TNF-α and IL-1β mRNA and corresponding increases in TNF-α and IL-1β proteins. Exposure of monocytes to glucan particles for 0–30 min and washing before continued incubation for 4 h in particle-free buffer induced production and secretion of TNF-α and IL-1β in a time-dependent fashion compatible with phagocytosis. The pretreatment of monocyte monolayers with trypsin reduced glucan-induced production of TNF-α and IL-1β in a dose-dependent manner with 5 μg/ml of trypsin effecting reductions of greater than 50%. Thus, glucan particles induce human monocyte production of TNF-α and IL-1β by a mechanism that is dependent on trypsin-sensitive β-glucan receptors.