The ability of calcium (Ca²⁺) to effect changes in growth cone motility requires remodeling of the actin cytoskeleton. To understand the mechanisms involved, we evaluated the effect of elevated intracellular calcium ([Ca²⁺]_i) on actin bundle dynamics, organization, and retrograde flow in the large growth cones of identified Helisoma neurons. Depolarization with 15 mm KCl (high K⁺) for 30 min caused a rapid and sustained increase in [Ca²⁺]_i and resulted in longer filopodia, shorter actin ribs, and a decrease in lamellipodia width. Time-lapse microscopy revealed that increasing [Ca²⁺]_i affected actin bundle dynamics differently at the proximal and distal ends. Filopodial lengthening resulted from assembly-driven elongation of actin bundles whereas actin rib shortening resulted from a distal shift in the location of breakage. Buckling of ribs occurred before breakage, suggesting nonuniform forces were applied to ribs before shortening. Calcium (Ca²⁺) influx also resulted in a decrease in density of F-actin in bundles, as determined by contrast changes in ribs imaged by differential interference contrast microscopy and fluorescent intensity changes in rhodamine-labeled ribs. The velocity of retrograde flow decreased by 50% after elevation of [Ca²⁺]_i. However, no significant change in retrograde flow occurred when the majority of changes in actin bundles were blocked by phalloidin. This suggests that inhibition of retrograde flow resulted from Ca²⁺-induced changes in the actin cytoskeleton. These results implicate Ca²⁺ as a regulator of actin dynamics and, as such, provide a mechanism by which Ca²⁺ can influence growth cone motility and behavior.