A sandwich ELISA was developed specific for human bactericidal/permeability-increasing protein (BPI), using Mg++ ions to abrogate disturbance by lipopolysaccharide of BPI measurement and to prevent aspecific adherence of BPI to solid phase. In fresh EDTA or heparinized plasma of healthy volunteers BPI was not detectable, whereas in serum BPI was present, indicating that coagulation activates polymorphonuclear leukocytes to release BPI. Furthermore, BPI was present in plasma of critically ill intensive care unit (ICU) patients, in bronchoalveolar lavage fluid of patients suspected of having pneumonia, in wound fluid, and in pleural fluid. In sub-groups of samples with culture-proven bacteria, mean BPI levels were increased compared with subgroups without bacteria, although the differences were only significant in EDTA plasma of ICU patients. These findings indicate the presence of BPI during pathologic conditions. The physiologic role of the released BPI has to be further elucidated.