The release of free cytosolic calcium is a secondary messenger for many cell functions. Here we study the coupling between the release of intracellular calcium and motility responses of the human neutrophil. Two groups of motility responses are studied: motility responses in the presence of adhesion, such as cell crawling and phagocytosis, and motility responses 'in suspension', such as pseudopod formation. The motility responses are stimulated by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the release of calcium is monitored by measuring the fluorescence from fluo-3. fMLP induces a single release of free cytosolic calcium both in suspended cells and in crawling cells. Calcium release is a threshold process where the number of cells releasing calcium is dependent on the chemoattractant concentration while the amount of released calcium is not. For suspended cells the threshold fMLP concentration for calcium release is in the order of 10(-7) M, while for crawling cells it is in the order of 5x10(-9) M. The smaller value of the threshold fMLP concentration for crawling cells compared to that for suspended cells suggests that bound adhesion receptors are involved in the calcium release. The threshold fMLP concentration for suspended cells is also larger than the minimum fMLP concentration (in the order of 10(-10) M) for initiating pseudopod formation. So, there is a range of fMLP concentrations where pseudopod formation occurs without calcium release. To explore this relationship further, pseudopod extension and calcium release are stimulated many times in a single cell by using fMLP concentrations above the threshold. The result is that calcium release is desensitized by fMLP while pseudopod extension is not. All the results taken together suggest that the release of free cytosolic calcium and the rearrangement of the F-actin network during motility follow different signaling pathways.