The influence of endothelial cells (ECs) on polymorphonuclear leukocyte (PMN) motility was examined using in vitro assays of PMN diapedesis and chemotaxis. ECs are seen to release arachidonic acid (20:4) metabolites that directly increase or decrease PMN movement, with their general effect being enhanced motility. This effect can be increased or decreased by treating ECs with stimulators or inhibitors of 20:4 metabolism, respectively. The metabolites include thromboxane B2, which increases PMN random motility, chemotaxis, and diapedesis in a dose- responsive manner and which acts as a chemoattractant; 6-keto-PGF1 alpha, which increases chemotaxis and diapedesis at high doses but decreases these responses at low doses; and a lipoxygenase-derived metabolite, suggested to be 5-hydroxyeicosatetraenoic acid, which increases chemotaxis and diapedesis. Thromboxane A2 and prostacyclin mimetics also affect chemotaxis in qualitatively similar manners as TxB2 and 6-keto-PGF1 alpha, respectively, but display greater potency. EC release of these metabolites is also seen to be substratum modulated, with an increased production by cells cultured on extracellular matrices. These results suggest that ECs are capable of modulating PMN motility and suggest a role for ECs in the control of PMN diapedesis.