The relationship between cytosolic free calcium concentration ([Ca2+]i) and human neutrophil motility was studied by video microscopy. Neutrophils stimulated by a uniform concentration of an N-formylated peptide chemoattractant (f-Met-Leu-Phe) were tracked during chemokinetic migration on albumin, fibronectin, and vitronectin. [Ca2+]i buffering with quin2 resulted in significant decreases in mean speed on albumin. To further characterize the relationship between [Ca2+]i changes and motility we carried out a cross-correlation analysis of [Ca2+]i with several motility parameters. Cross-correlations between [Ca2+]i and each cell's speed, angle changes, turn strength, and persistent forward motion revealed (i) a positive correlation between [Ca2+]i and cell speed (p < 0.05), (ii) no significant correlation between turns and calcium spikes, and (iii) the occurrence of turns during periods of low speed. Significant negative correlations between [Ca2+]i and angle change were noted on the high adhesion substrates vitronectin and fibronectin but not on the low adhesion substrate albumin. These data imply that there is a general temporal relationship between [Ca2+]i, speed, and persistent motion. However, the correlations are not sufficiently strong to imply that changes in [Ca2+]i are required proximal signals for velocity changes.