Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.