We demonstrated that rat peritoneal cells synthesize, and release into the culture medium, substances that elicit a chemotactic responsiveness of rat macrophages. These substances were identified by HPLC analysis as two diastereoisomers of LTB4, precisely delta-6-trans-LTB4 and delta-6-epi-trans-LTB4. Peritoneal cells release, also, other lipoxygenase metabolites such as LTD4 and LTB4, which were shown not to be active in eliciting chemotaxis of rat macrophages, in agreement with data of other authors. Quantitative assays for the measurement of PMNs or macrophage chemotaxis were carried out in Boyden chambers. Release of leukotrienes by rat peritoneal cells into the culture medium was strongly enhanced upon stimulation with calcium ionophore A23187, an agent known to increase cytoplasmic Ca2+ concentration, thus leading to the activation of Ca2+-dependent phospholipase and a consequent increase in the amount of endogenous free arachidonic acid available for biotransformation. The action of several inhibitors of arachidonate metabolism at concentrations more selective for the lipoxygenase than the cycloxygenase pathway, was also studied. alpha-Tocopherol and ASA were shown to be the most selective in inhibiting the synthesis of both the LTB4 diasteroisomers active as enhancers of rat macrophage chemotaxis.