The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70 s6k ) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70 s6k activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70 s6k . Furthermore, the p110α form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70 s6k , as well as to a golgi-like distribution. In contrast, PI 3-kinase p110γ colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110α, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110α and p110α-associated PI 3-kinase activity, confirming the presence of a p85-free p110α pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110α in the stimulation by thrombin of p70 s6k activation and actin stress fiber formation.