Recent investigations postulate an endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen (HK) on HUVEC, PK is activated to kallikrein. PKA is found in the 15,800 X g pellet of HUVEC lysates using an assay that measures PK activation only when bound to HK linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography result in 4 protein bands on SDS-PAGE. One protein in the 73 kDa band is identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity is a single homogenous peak identical in migration to the 73 kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA is blocked by DFP (1 mM), PMSF (3 mM), leupeptin (100 microM) antipain (IC50=2 microM), HgCl2 (IC50=500 microM), Z-Pro-Pro-aldehyde-dimethyl acetate (IC50=1 microM), and corn trypsin inhibitor (IC50=40 nM). PKA does not correct the coagulant defect in factor XII deficient plasma, is purified from HUVEC cultured in factor XII deficient serum, is not detected by antibody to factor XII, does not activate FXI, and is not inhibited by a neutralizing antibody to FXII. Angiotensin II (IC50=2 microM) or bradykinin (IC50=100 microM), but not angiotensin II1-7 or bradykinin1-5, and the prolyl oligopeptidase inhibitor, Fmoc-Ala-Pyr-CN (IC50=50 nM), also block purified PKA activation of PK. The Km of PK activation by PRCP is 6.7 nM. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HUVEC-associated PK activator.