Treatment of human airway smooth muscle (HASM) with interleukin (IL)-1β alone or in combination with serum or growth factors is known to induce cyclooxygenase (COX)-2 and prostaglandin E2 synthesis, an effect presumably underlying IL-1β–mediated β2 adrenergic receptor and epoprostanoid receptor desensitization and inhibition of mitogen-stimulated cell growth in HASM. Although COX-2 induction is clearly required and COX-2 activity is believed to be limiting in IL-1β-stimulated prostaglandin E2 synthesis, the role of phospholipase A2 (PLA2) in this process in unclear. We therefore assessed potential alterations in the following: (1) cytosolic PLA2 (cPLA2) protein levels, (2) cPLA2 phosphorylation state, and (3) PLA2 activity in growth-arrested HASM cultures treated with IL-1β or growth factors. Treatment with IL-1β induced a time-dependent increase in cPLA2 protein levels (assessed by immunoblotting) with a twofold increase observed at 18 h. Epidermal growth factor had only a minimal effect on cPLA2 induction, but cotreatment with IL-1β plus endothelial growth factor was synergistic, resulting in a greater than fourfold induction. cPLA2 induction was partially inhibited by the p38 inhibitor SB203580 and the classical protein kinase C inhibitor Bis I. IL-1β induced a rapid phosphorylation of cPLA2, which peaked at approximately 1 h and then waned toward basal levels by 18 h. Lastly, PLA2 activity in cultured HASM, characterized by cleavage of a phospholipid analog conjugated to a BODIPY fluorophore (D3803; Molecular Probes; Eugene, OR), was regulated by IL-1β but occurred against a background of high basal PLA2 activity in cell culture medium, suggesting an important role for secretory PLA2 in HASM arachidonate metabolism. These data suggest that regulation of HASM PLA2 activity may be important in cytokine- and growth factor-mediated eicosanoid synthesis and associated functional consequences.