Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca2+. The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP3 receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP2 levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca2+.