In trauma or sepsis, monocytes and macrophages release mediators such as tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2). Although patients may be exposed to more than one stimulus, the effect of repetitive endotoxin (LPS) stimulation on human monocytes is poorly characterized. Human peripheral blood monocytes obtained from healthy volunteers were pretreated with endotoxin (LPS1) for 24 h. Cultures were then restimulated for 24 h with a second, activating LPS stimulus (LPS2) at various concentrations and supernatant mediators (TNF, IL-1, IL-6, and PGE2) measured. Serum cytokine levels of normal monocyte donors were compared to basal and LPS-stimulated cytokine release of their monocytes in vitro. LPS2 increased all mediators in a dose-dependent manner in the absence of LPS1 pretreatment. LPS1 significantly increased LPS2-triggered monocyte secretion of IL-1, IL-6, and PGE2, but inhibited TNF release. Cell-associated TNF and IL-1 were also inhibited and enhanced in parallel with supernatant levels of the respective cytokines. Serum cytokine levels were low, showed wide variation, and correlated poorly with in vitro LPS-triggered cytokine production. Human monocyte mediator production is differentially regulated by endotoxin pretreatment. Provocative in vitro testing of monocytes could identify prior LPS exposure and may be more useful than serum cytokine measurements.