We found exaggerated chemotaxis in plasma treated with EDTA and thought that the EDTA might itself be inhibiting a tonic inhibitor(s) of chemotaxis. Our plasma fractionations suggested that evidence should be sought for a lipid moiety carrying this activity, and on spectrometry (LC-MS-MS together with GC-MS analyses), the biologically active but not the inactive fraction contained oleic and arachidonic acids. Because fatty acids are largely protein bound, we flooded plasma preparations with delipidated albumin, reasoning that it would bind enough fatty acids, including inhibitory ones, to counter their tonic inhibition. Indeed, we observed dramatic increases in chemotaxis. Hence, adding delipidated albumin to plasma has a similar effect to that of adding EDTA-amplification of the chemotactic response. Oleic acid in physiologic concentrations diminishes the magnifying effects of both EDTA and of delipidated albumin, and in fact diminishes the chemotactic response even without the presence of the amplifiers of chemotaxis. In contrast, arachidonic acid amplifies further the effect of EDTA but not of delipidated albumin, and this augmentation appears to be caused by an EDTA-dependent enrichment of the chemotactic gradient with leukotriene B4 (LTB4). We conclude that oleic acid, the blood levels of which vary among individuals, is at least one tonic inhibitor of chemotaxis in plasma.fatty acids ͉ oleic acid ͉ neutrophils T his work began with the videomicroscopic observation that polymorphonuclear leukocytes (PMNs) from blood anticoagulated with EDTA, but not with heparin, segregate into groups, usually around a monocyte (1, 2). The EDTA effect occurred in serum as well as in plasma (2). When the monocytes became less adhesive and moved from the center of the field, the PMNs did not follow. Rather, they ignored the monocyte, remaining focused on the place where it used to be, and often continuing to arrive in large numbers; they appeared to have taken over the generation of a chemotactic gradient, replacing the one initially provided by the adherent monocyte (2). Moreover, this prolonged chemoattraction did not depend on triggering by monocytes; we saw large aggregates accrue around PMNs containing ingested material, damaged (motionless, mummified appearance) PMNs, or detritus in the slide preparation.To pursue the phenomenon of prolonged chemotaxis in EDTA/ plasma in a more systematic approach, we used the chemoattractant gradient established by an erythrocyte destroyed by laser microirradiation (3, 4). With videomicroscopy, one can observe directly and continuously the behavior of PMN in real time before, during, and after establishment of a chemotactic gradient. Within several seconds of the laser flash, PMNs in the area turn in the direction of the newly created chemotactic target. PMNs then move to the target, and surround it for several minutes, before departing cells begin to outnumber arriving ones, presumably mirroring a decline in the exudation of chemoattractant. As in the case of chemotactic gradients ini...