Protease nexin 1 (PN-1) is a protease inhibitor secreted by cultured fibroblasts that forms complexes with certain serine proteases; the complexes bind back to the cells and are internalized and degraded. In the present studies, a panel of PN-1 monoclonal antibodies (mAbs) was isolated; none showed detectable cross-reactivity with four related plasma protease inhibitors. Four purified mAbs (mAbp1, mAbp6, mAbp9, and mAbp18) were tested for their ability to block the formation of complexes between PN-1 and target proteases. mAbp1, as well as a rabbit polyclonal anti-PN-1 IgG preparation, did not block formation of 125I-thrombin-PN-1 complexes. mAbp6, mAbp9, and mAbp18 blocked the formation of 125I-thrombin-PN-1 and 125I-urokinase-PN-1 complexes at stoichiometric concentrations of mAb and PN-1. Studies on their ability to block formation of 125I-trypsin-PN-1 complexes showed that mAbp18 also blocked this reaction at stoichiometric concentrations with PN-1 whereas mAbp6 and mAbp9 blocked less effectively. Thus, mAbp18 appears to bind at or close to the reactive center of PN-1. The blocking mAbs should be useful in studies to probe physiological functions of PN-1.