Secretory nonpancreatic group IIA phospholipase A2 (sPLA2), a lipolytic enzyme found in plasma, is thought to play an important role in inflammation. In patients with sepsis, a strong positive correlation is observed between the plasma level of sPLA2 and poor clinical outcome in sepsis. We have thus asked whether sPLA2 could play a role in enabling responses of cells to bacterial lipopolysaccharide (LPS), a key contributor to sepsis. In the presence of sPLA2, cellular responses to LPS were significantly increased. This was demonstrated in assays of LPS-stimulated interleukin-6 (IL-6) production in whole blood and binding of freshly isolated human polymorphonuclear neutrophils (PMN) to fibrinogen-coated surfaces. We further found that sPLA2 enhanced binding of labeled LPS to PMN, and that the sPLA2-mediated cell responses to LPS were all blocked by monoclonal antibodies directed against membrane CD14. Two properties ofsPLA2 may contribute to its activity to mediate responses to LPS. sPLA2 appears to bind LPS because pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholid substrate, and incubation of sPLA2 with BODIPY-LPS micelles resulted in enhanced fluorescence, presumably from the disaggregation of the LPS aggregates. Additional studies demonstrated that the esterolytic function of sPLA2 is also needed both for the disaggregation of LPS and CD14-dependent cell stimulation. The precise mechanisms by which LPS-binding and esterolytic activity contribute to sPLA2 activity are not clear but our data strongly suggest that these activities result in interaction of LPS with CD14 and subsequent cell activation.