We have investigated cellular Ca²⁺ regulation during A2058 human melanoma cell chemotaxis to type IV collagen (CIV). We have identified ₂₁-integrin as the primary mediator of A2058 cell response to CIV in vitro. Integrin ligation initiated a characteristic intracellular Ca²⁺ concentration ([Ca²⁺]_i) response consisting of an internal release and a receptor-mediated Ca²⁺ entry. Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible internal Ca²⁺ stores while initiating a store-operated Ca²⁺ release (SOCR). CIV-mediated Ca²⁺ entry was additive to TG-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca²⁺ initiated a sustained influx. Elevated [Ca²⁺]_i from TG-SOCR or ionophore significantly attenuated cell migration to CIV by recruiting the Ca²⁺/calcineurin-mediated signaling pathway. Furthermore, low [Ca²⁺]_i induced by EGTA application in the presence of ionophore fully restored cell motility to CIV. Together, these results suggest that [Ca²⁺]_i signaling accompanying A2058 cell response to ₂₁-integrin ligation is neither necessary nor sufficient and that elevated [Ca²⁺]_i downregulates cell motility via a calcineurin-mediated mechanism in A2058 cell chemotaxis to CIV.