Idiopathic myelofibrosis (IM) is characterized by increased numbers of CD34(+) cells in the peripheral blood (PB). We explored the possible mechanisms underlying this abnormal trafficking of CD34(+) cells. Plasma levels of neutrophil elastase (NE), total and active matrix metalloproteinase 9 (MMP-9), and soluble vascular cell adhesion molecule-1 (sVCAM-1) were dramatically increased in IM. The absolute number of CD34(+) cells in the PB was correlated with the levels of sVCAM-1. Marked elevations of the levels of NE but not total and active MMP-9 as well as MMP-2 were detected in media conditioned by IM mononuclear cells (MNCs) as compared with that of healthy volunteers. IM MNC-conditioned media, however, was shown by zymographic analysis to contain increased gelatinolytic activity corresponding to the molecular weight of MMP-9. IM MNC-conditioned media also exhibited a greater ability to cleave VCAM-1 and c-kit in vitro, consistent with the biologic actions of NE. In addition, the increased ability of IM PB CD34(+) cells to migrate through a reconstituted basement membrane was diminished by several inhibitors of MMP-9 activity, indicating that these cells express increased levels of this MMP. These data indicate that a proteolytic environment exists in IM which might result in the sustained mobilization of CD34(+) cells.