Divalent cations are thought essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the effects of 10 mM EDTA on locomotion of human blood polymorphonuclear leukocytes (PMN). In very thin slide preparations, EDTA did not impair either random locomotion or chemotaxis; motile behavior appeared to benefit from the close approximation of slide and coverslip (''chimneying''). In preparations twice as thick, PMN in EDTA first exhibited active deformability with little or no displacement, then rounded up and became motionless. However, on creation of a chemotactic gradient, the same cells were able to orient and make their way to the target, often, however, losing momentarily their purchase on the substrate. In either of these preparations without EDTA, specific antibodies to 2 integrins did not prevent random locomotion or chemotaxis, even when we added antibodies to 1 and ␣v3 integrins and to integrin-associated protein, and none of these antibodies added anything to the effects of EDTA. In the more turbulent environment of even more media, effects of anti-2 integrins became evident: PMN still could locomote but adhered to substrate largely by their uropods and by uropod-associated filaments. We relate these findings to the reported independence from integrins of PMN in certain experimental and disease states. Moreover, we suggest that PMN locomotion in close quarters is not only integrin-independent, but independent of external divalent cations as well.Divalent cations are thought to be essential for motile function of leukocytes in general, and specifically for the function of adhesion molecules critical to a number of their motile activities (1, 2). In examining effects of concentrations of EDTA in the anticoagulant range (Ն1.8 mM) for other reasons, we noted that polymorphonuclear leukocytes (PMN) in warmed sealed slide preparations were not impaired in either random locomotion or chemotaxis. In the current study we have pushed the concentration of EDTA to a point (10 mM) where [Ca 2ϩ ] i or [Mg 2ϩ ] i in the medium would be ϽnM, with similar results.In the slide preparations, a chemotactic gradient is created by the targeted destruction of one or a few erythrocytes resulting from a brief laser flash (3). We examine the behavior of PMN under direct microscopic observation with concomitant time-lapse video recording. Hence, we can follow directly and continuously the orientation and trajectory of PMN before, during, and after establishment of a chemotactic gradient (4).In a sense, the results with EDTA make additional work on specific adhesion molecules redundant, as the function of all of them is thought to be divalent-cation-dependent. However, an examination of relevant leukocyte integrins through the use of mAbs was of interest so that their effects in this system, or lack of effects, might be placed in context wit...