Abstract. During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front.The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca 2+ signal may trigger the cytoskeletal reorganiTation requlred for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca 2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca 2+ signals. To identify these messengers, Ca 2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]t increased rapidly from ~100 nM to >.~400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of '~ 200 nM.The initial Ca 2+ spike was insensitive acutely to removal of extracellular Ca 2+ but was abolished by treatments expected to deplete internal Ca 2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lametlipod activity and suppressed the Ca 2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca 2+ spike on stimulation. Although cell flattening and larneUipod activity were induced by chemoattractant when the normal Ca 2+ response was blocked, cells failed to polarize and crawl, indicating that Ca 2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca 2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca 2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.D RECTIONAL persistence during locomotion in a stimulus gradient and rapid reorientation when the direction of the stimulus gradient changes are virtually certain to involve a complex web of highly interactive intracellular processes that stabilize directionality in a way that can be immediately and effectively overridden. Although components of the signal transduction pathways that control cell orientation have been identified in many types of cells, little is known about the mechanisms involved in translating the spatio-temporal patterns of the stimulus into appropriate changes in intracellular organization. Ca2+-sensitivity of a large number of cytoskeletal proteins suggests that Ca 2+ could play an important role. Thus considerable effort has been devoted to characteri...

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