The β2-integrins on leukocytes can serve as a signaling unit during cell adhesion and locomotion, and to further clarify this important property we investigated the possible mechanisms of β2-integrin-induced activation of PtdIns 3-kinase. It has previously been demonstrated that clustering of β2-integrins activates p21ras by a tyrosine kinase-dependent pathway, and here we show that active p21ras interacts with its downstream target, PtdIns 3-kinase. Engagement of β2-integrins also activates the tyrosine kinases p58c-fgr and p59/61hck and causes them to associate with the p85 subunit of PtdIns 3-kinase. These findings suggest a mechanism whereby p58c-fgr and p59/61hck are directly involved in the activation of PtdIns 3-kinase. No coupling between p58c-fgr and p59/61hck could be detected; hence these kinases probably trigger independent but parallel signals to PtdIns 3-kinase. The effect of β2-integrin clustering on PtdIns 3-kinase activity was monitored as the activation of protein kinase B (PKB). Stimulation of PKB by β2-integrins was abolished by genistein and wortmannin but not by using methyl transferase inhibitors to abrogate the influence of p21ras-related proteins. Thus, even if PtdIns 3-kinase is not activated by p21ras, it can maintain full enzyme activity due to the mentioned interaction with p58c-fgr or p59/61hck. These tyrosine kinases apparently activate similar pathways that operate in parallel and therefore have the potential to substitute for each other in mediating adhesion and regulating cell locomotion.