Background: Matrix metalloproteinase (MMP)-9 levels are increased in bronchoalveolar lavage (BAL) fluid from patients with severe asthma on high doses of glucocorticoids (GCs). Objective: We sought to identify neutrophils as the source of increased BAL fluid MMP-9 in severe asthma and to evaluate the effects of GCs on this MMP-9. Methods: MMP-9 protein, activity, and mRNA were measured in BAL fluid and cells at baseline, and after in vitro GCs in patients with severe asthma and controls using enzyme immunoassays, zymography. Western blotting, and real-time PCR. Results: The high molecular weight (HMW) form of MMP-9 was significantly increased in severe asthma (P = .02). Western blotting confirmed a heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin. The HMW MMP-9 correlated with BAL neutrophils (r = .65, P < .0001). BAL cell supernatant MMP-9 protein levels also tended to be higher in patients with severe asthma (overall, P = .09), whereas the HMW activity form was increased (P = .03). MMP-9 protein (and HMW activity) correlated with neutrophils in the cell pellet (r = .75, P < .0001). In contrast to protein and activity, BAL cell mRNA levels were marginally lower in patients with severe asthma than in control subjects (overall, P = .06). Although GCs decreased BAL cell MMP-9 protein and mRNA in vitro, the effect was significantly smaller in severe asthma (P < .01 for both). GCs decreased the pro-MMP-9 activity in patients with severe asthma and normal control subjects, while, having no effects on the HMW form (P = .22). Peripheral blood neutrophil MMP-9 protein was not affected by GCs. Conclusions: BAL neutrophils contribute to BAL fluid MMP-9 protein and activity and are poorly inhibited by GCs.