Aequorin luminescence has been utilized to determine the spatial and temporal fluctuations of the free calcium ion concentration [Ca"] in Chaos carolinensis during ameboid movement, pinocytosis, and capping. The [Ca"] increases above _10-7 M during normal ameboid movement . Three types of luminescent signals are detected in cells: continuous luminescence, spontaneous pulses, and stimulated pulses . Continuous luminescence is localized in the tails of actively motile cells, and spontaneous pulses occur primarily over the anterior regions of cells. We are sometimes able to correlate the spontaneous pulses with extending pseudopods, whereas stimulated pulses are induced by mechanical damage, electrical stimulation, concanavalin A-induced capping, and pinocytosis. The localization of both distinct actin structures and sites where [Ca"] increases suggests cellular sites of contractile activity . The independent evidence from localizing actin structures and the distribution of [Ca"] can also be viewed in relation to the solation-contraction coupling hypothesis defined in vitro.Calcium regulation of cytoplasmic structure and contractility in ameboid cells was first identified in single cell models from Chaos carolinensis (66) . The calcium regulation of both gelation and contraction was subsequently described in bulk cell-free extracts (21, 68, 69), in partially reconstituted models (31), and in single living cells (70) . Recently, a high-affinity calciumbinding protein which regulates gelation and plays a role in regulating contraction (77) (see references 73 and 74 for reviews) has been isolated from Dictyostelium discoideum . Evidence for calcium regulation of cytoplasmic structure and/or contractility has also been demonstrated in cell models from other ameboid cells, including Acanthamoeba (50), Ehrlich tumor cells (45), and macrophages (78). In addition, a great deal of indirect evidence has also supported the suggestion that calcium regulates at least some of the events involved in ameboid movements (see references 33 and 74 for reviews) .A direct identification of fluctuations in the free calcium ion concentration in living cells is necessary to identify calcium as a physiologically relevant intracellular messenger involved in cell motility . This is especially true because other parameters, including changes in pH, can also affect cytoplasmic structure THE JOURNAL of CELT BIOLOGY -VOLUmE 86 AUGUST 1980 599-607 ©The Rockefeller University Press -0021-9525/80/08/0599/09 $1 .00 and contractility in vitro (21, 68, 69).' Ultimately, a map of the spatial and temporal distribution of all potential regulatory factors must be constructed and correlated with motile behavior in living cells.Aequorin luminescence (9-11, 56, 60, 61) has been used as a highly sensitive indicator of intracellular free calcium in several types of cells (see references 9, 10 and 56 for reviews) . The high signal to noise ratio, relative specificity for Ca", lack of toxicity, and ease of signal detection have made this technique attr...