The precise mechanisms that regulate increases in airway smooth muscle (ASM) mass in asthma are unknown. This study determined whether class IA phosphatidylinositol 3-kinase (PI3K) is sufficient to stimulate DNA synthesis and characterized the PI3K isoforms expressed in human ASM cells. ASM cells express class IA, II, and III PI3K but not class IB. Because thrombin induces ASM cell proliferation, we investigated whether thrombin can stimulate class IA PI3K. Transient transfection of ASM cells with hemagglutinin-tagged p85 PI3K followed by immunostaining revealed that in quiescent cells, p85 was expressed diffusely in the cytoplasm and after stimulation with thrombin p85 translocated to the cell membrane. Microinjection of ASM cells with a dominant negative class IA PI3K inhibited thrombin-induced DNA synthesis by 30% and epidermal growth factor (EGF)- or serum-induced DNA synthesis by 13 and 28%, respectively (P < 0.05 by chi(2) analysis). In parallel experiments, transfection or microinjection of cells with constitutively active PI3K markedly increased DNA synthesis in transfected cells 10.5-fold and in microinjected cells 12.7-fold (P < 0.05 by chi(2) analysis) compared with cells transfected or microinjected with control plasmid. Interestingly, constitutively active PI3K augmented EGF-induced DNA synthesis but had little effect on that induced by serum or thrombin in ASM cells. Collectively, these data suggest that class IA PI3K is activated by thrombin and is sufficient to induce ASM cell growth.