Purified human monocytes release and metabolize endogenous arachidonic acid (20:4) from phospholipid stores when challenged with particulate inflammatory stimuli or the calcium ionophore A23187. Using radiolabeled cultures, the percentage of total [3H]20:4 released was similar with each type of stimulus. However, the spectrum of 20:4 metabolites differed. With opsonized zymosan (OpZ) or Sephadex beads coated with IgG immune complexes (Ig-beads), the predominant product was thromboxane (25% of the total) together with smaller amounts of other cyclo-oxygenase products and lipoxygenase metabolites. Levels of thromboxane synthesis by monocytes were comparable to those by platelets, as measured by radioimmunoassay. In contrast, exposure to the nonspecific agent A23187 led to mainly lipoxygenase products (70% of the total). Monocytes isolated from mononuclear cell fractions of peripheral blood contain platelets specifically rosetted to their surfaces. These platelet contaminants were removed by sequential incubations of monocytes in serum and EDTA followed by adherence and detachment from tissue culture vessels. The presence of platelets in routinely isolated monocytes presented a major difficulty in the study of human monocyte 20:4 metabolism since platelets also synthesize thromboxane. Loss of 12-HETE synthesis (16-fold reduction relative to 5-HETE) in A23187-stimulated cultures provided a convenient measure of platelet depletion. This together with the response to monocyte-specific stimuli (OpZ and Ig-beads) allowed for the distinction between monocyte and platelet 20:4 metabolism.